Anti-addl antibodies and uses thereof

ABSTRACT

The present invention relates to antibodies that differentially recognize multi-dimensional conformations of Aβ-derived diffusible ligands, also known as ADDLs. The antibodies of the invention can distinguish between Alzheimer&#39;s Disease and control human brain extracts and are useful in methods of detecting ADDLs and diagnosing Alzheimer&#39;s Disease. The present antibodies also block binding of ADDLs to neurons, assembly of ADDLs, and tauphosphorylation and are there useful in methods for the preventing and treating diseases associated with soluble oligomers of amyloid β 1-42.

INTRODUCTION

This application claims the benefit of priority from U.S. provisionalpatent application Ser. Nos. 60/621,776, filed Oct. 25, 2004,60/652,538, filed Feb. 14, 2005, 60/695,526 filed Jun. 30, 2005 and60/695,528 filed Jun. 30, 2005 whose contents are incorporated herein byreference in their entireties.

This invention was made in the course of research sponsored, in part, bythe National Institutes of Health (Grant Nos. NIH RO1-AG18877 and NIHRO1-AG22547). The U.S. government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Alzheimer's Disease is a progressive and degenerative dementia (Terry,et al. (1991) Ann. Neurol. 30:572-580; Coyle (1987) In: Encyclopedia ofNeuroscience, Adelman (ed.), Birkhäuser, Boston-Basel-Stuttgart, pp29-31). In its early stages, Alzheimer's Disease manifests primarily asa profound inability to form new memories (Selkoe (2002) Science298:789-791), reportedly due to neurotoxins derived from amyloid beta(Aβ). Aβ is an amphipathic peptide whose abundance is increased bymutations and risk factors linked to Alzheimer's Disease. Fibrils formedfrom Aβ constitute the core of amyloid plaques, which are hallmarks ofan Alzheimer's Disease brain. Analogous fibrils generated in vitro arelethal to cultured brain neurons. These findings indicate that memoryloss is a consequence of neuron death caused by fibrillar Aβ.

Despite strong experimental support for fibrillar Aβ and memory loss, apoor correlation exists between dementia and amyloid plaque burden(Katzman (1988) Ann. Neurol. 23:138-144). Moreover, transgenic hAPP mice(Dodart, et al. (2002) Nat. Neurosci. 5:452-457; Kotilinek, et al.(2002) J. Neurosci. 22:6331-6335), which develop age-dependent amyloidplaques and, most importantly, age-dependent memory dysfunction, showthat within 24 hours of vaccination with monoclonal antibodies againstAβ memory loss can be reversed with no change in plaque levels. Suchfindings are not consistent with a mechanism for memory loss dependenton neuron death caused by amyloid fibrils.

Additional neurologically active molecules formed by Aβ self-assemblyhave been suggested. These molecules include soluble Aβ oligomers, alsoreferred to as Aβ-derived diffusible ligands or ADDLs. Oligomers aremetastable and form at low concentrations of Aβ1-42 (Lambert, et al.(1998) Proc. Natl. Acad. Sci. USA 95:6448-6453). Aβ oligomers rapidlyinhibit long-term potentiation (LTP), a classic experimental paradigmfor memory and synaptic plasticity. As such, memory loss stems fromsynapse failure, prior to neuron death and synapse failure by Aβoligomers, not fibrils (Hardy & Selkoe (2002) Science 297:353-356).Soluble oligomers have been found in brain tissue and are strikinglyelevated in Alzheimer's Disease (Kayed, et al. (2003) Science300:486-489; Gong, et al. (2003) Proc. Natl. Acad. Sci. USA100:10417-10422) and in hAPP transgenic mice Alzheimer's Disease models(Kotilinek, et al. (2002) J. Neurosci. 22:6331-6335; Chang, et al.(2003) J. Mol. Neurosci. 20:305-313).

A variety of Alzheimer's Disease treatment options have been suggested.Vaccine clinical trials have revealed that persons mounting a vigorousimmune response to the vaccine exhibit cognitive benefit (Hock, et al.(2003) Neuron 38:547-554); however, frequency of CNS inflammation causedearly termination of part of the trial (Birmingham & Frantz (2002) Nat.Med. 8: 199-200). As an alternative to a vaccine, therapeutic antibodiesthat target ADDLs without binding monomers or fibrils have beensuggested (Klein (2002) Neurochem. Int. 41:345-352). ADDLs are highlyantigenic, generating oligomer-selective polyclonal antibodies inrabbits at concentration of ˜50 μg/mL (Lambert, et al. (2001) J.Neurochem. 79:595-605). Results from transgenic mice models also suggestthat antibodies can be successful in reversing memory decline (Dodart,et al. (2002) Nat. Neurosci. 5:452-457). Accordingly, there is a need inthe art for ADDL-selective therapeutic antibodies for the prevention andtreatment of Alzheimer's Disease. The present invention meets this need.

SUMMARY OF THE INVENTION

The present invention is an isolated antibody, or fragment thereof,capable of differentially recognizing a multi-dimensional conformationof one or more Aβ-derived diffusible ligands. In particular embodiments,the antibody of the present invention is in admixture with apharmaceutically acceptable carrier. In other embodiments, the antibodyof the present invention is in a kit.

Methods for preventing binding of Aβ-derived diffusible ligands to aneuron, inhibiting assembly of Aβ-derived diffusible ligands, andblocking the phosphorylation of tau protein at Ser202/Thr205 employingan antibody or antibody fragment which binds a multi-dimensionalconformation of one or more Aβ-derived diffusible ligands are alsoprovided.

The present invention further embraces a method for prophylactically ortherapeutically treating a disease associated with Aβ-derived diffusibleligands using an antibody of the instant invention. Administration of anantibody of the invention can prevent binding of Aβ-derived diffusibleligands to a neuron thereby preventing or treating the diseaseassociated with Aβ-derived diffusible ligands.

The present invention is also a method for identifying a therapeuticagent that prevents the binding of Aβ-derived diffusible ligands to aneuron. This method of the invention involves contacting a neuron withAβ-derived diffusible ligands in the presence of an agent and using anantibody of the present invention to determine binding of the Aβ-deriveddiffusible ligands to the neuron in the presence of the agent.

The present invention also embraces a method for detecting Aβ-deriveddiffusible ligands in a sample and a method for diagnosing a diseaseassociated with Aβ-derived diffusible ligands. Such methods involvecontacting a sample with an antibody of the instant invention so thatthe Aβ-derived diffusible ligands can be detected and a diseaseassociated with Aβ-derived diffusible ligands can be diagnosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results from an alkaline phosphatase assay, whereinanti-ADDL antibodies differentially block neurons.

FIG. 2 shows a summary of bADDL binding when B103 cells arepre-incubated with anti-ADDL antibodies.

FIG. 3 shows a summary of binding characteristics of antibodies capableof differentially recognizing multidimensional conformations of ADDLs.

FIG. 4 shows a summary of ADDL assembly inhibition of the antibodiesdisclosed herein.

FIG. 5 shows an N2A binding:k_(ADDL) correlation plot.

FIG. 6 shows the nucleic acid sequences for the heavy and light chainvariable regions, respectively, for murine anti-ADDL antibodies, 20C2(FIGS. 6A and 6B), 5F10 (FIGS. 6C and 6D), 2D6 (FIGS. 6E and 6F), 2B4(FIGS. 6G and 6H), 4E2 (FIGS. 6I and 6J), 2H4 (FIGS. 6K and 6L), 2A10(FIGS. 6M and 6N), 3B3 (FIGS. 6O and 6P), 1F6 (FIGS. 6Q and 6R), 1F4(FIGS. 6S and 6T), 2E12 (FIGS. 6U and 6V) and 4C2 (FIGS. 6W and 6X).Lower case letters indicate the antibody leader sequences and uppercaseletters indicate antibody variable region sequences. The nucleotidescoding for the complementary determining regions (CDRs) are underlined.

FIG. 7 shows comparisons of CDR1 (FIG. 7A), CDR2 (FIG. 7B), CDR3 (FIG.7C) sequences for the heavy chain variable regions and CDR1 (FIG. 7D),CDR2 (FIG. 7E), CDR3 (FIG. 7F) sequences for the light chain variableregions for the mouse anti-ADDL antibodies.

FIG. 8 shows the amino acid sequences for the heavy and light chainvariable regions, respectively, for humanized anti-ADDL antibodies 20C2(FIGS. 8A and 8B), 26D6 (FIGS. 8C and 8D), 4E2 (FIGS. 8E and 8F), 3B3(FIGS. 8G and 8H), 2H4 (FIGS. 8I and 8J) and 1F6 (FIG. 8K) created byCDR grafting. Sequences are presented as comparisons between the mousesequence, the most homologous human sequence obtained from the NCBIprotein database, the most homologous human genomic sequence and thehumanized sequence. Amino acids in the mouse, human and human genomicsequences that differ from the humanized sequences are in bold. CDRs areunderlined. Residues important for the maintenance of CDR loopconformation are indicated with an *. Conserved residues found at theVL/VH interface are indicated with a #. Potential glycosylation sitesare indicated by italic. For the 20C2 heavy chain two humanizedsequences were generated (HCVRA and HCVRB) that differ by one amino acidat position 24. In 20C2 HCVRA the human amino acid was used and in 20C2HCVRB the mouse amino acid was used. No light chain was designed for 1F6because it has the same sequence as that of the light chain for 4E2.

FIG. 9 shows the amino acid sequences for the heavy and light chainvariable regions, respectively, for humanized anti-ADDL antibodies 20C2(FIGS. 9A and 9B) and 26D6 (FIGS. 9C and 9D) created by veneering.Sequences are presented as comparisons between the mouse sequence, themost homologous human sequence obtained from the NCBI protein database,the most homologous human genomic sequence and the humanized sequence.Amino Acids in the mouse, human and human genomic sequences that differfrom the humanized sequences are bold. CDRs are underlined. Residuesimportant for the maintenance of CDR loop conformation are indicatedwith an asterisk. Conserved residues found at the VL/VH interface areindicated with a pound symbol. Potential glycosylation sites areindicated by italic. For the 20C2 heavy chain, two humanized sequenceswere generated (HCVRVenA and HCVRVenB) that differ by one amino acid atposition 81. In 20C2 HCVRVenA, the mouse amino acid was used and in 20C2HCVRVenB, the human amino acid was used. For the 26D6 heavy chain, threehumanized sequences were designed based on veneering (HCVR Ven1, Ven 2and Ven3) that differ at amino acids 11, 23, 15, 81, 89 and 118. In HCVRVen1, the mouse amino acid was used at all positions. In Ven2, the mouseamino acid was used for residues 81 and 118 and the human amino acid forresidues 11, 13, 15, and 89. In Ven3, the human amino acids were used atall positions. For the 26D6 light chain, two veneered humanizedsequences were designed (LCVR Ven1 and Ven2) that differ at amino acids88 and 105. In LCVR Ven1, the mouse amino acid was used at bothpositions and in Ven2, the human amino acid was used.

FIG. 10 shows nucleic acid sequences for the heavy and light chainvariable regions (HCVRs and LCVRs, respectively) for humanized anti-ADDLantibodies. CDR grafted HCVRs and LCVRs for 20C2, 2D6, 4E2, 3B3, 2H4,and IF6, are respectively presented in FIG. 10A to FIG. 10K. VeneeredHCVRs (VenA and VenB) and the LCVR for 20C2 are presented in FIG. 10L toFIG. 10N, whereas the veneered HCVRs (Ven1, Ven2, Ven3) and LCVRs (Ven1,Ven2) for 26D6 are presented in FIG. 10O to FIG. 10S. Uppercaseindicates antibody variable region sequences. CDRs are underlined.Variable region sequences were cloned into full heavy and light chainantibody expression vectors.

FIG. 11 shows the amino acid sequences for the full IgG1 and IgG2 m4humanized heavy chains and humanized Kappa light chains for anti-ADDLantibodies. FIG. 11A, CDR grafted 20C2 HCVRA IgG1; FIG. 11B, CDR grafted20C2 HCVRB IgG1; FIG. 11C, CDR grafted 20C2 HCVRA IgG2 m4; FIG. 11D, CDRgrafted 20C2 HCVRB IgG2 m4; FIG. 11E, CDR grafted 20C2 LCVR Kappa; FIG.11F, CDR grafted 26D6 HCVR IgG1; FIG. 11G, CDR grafted 26D6 HCVR IgG2m4; FIG. 11H, CDR grafted 26D6 LCVR Kappa; FIG. 11I, CDR grafted 4E2HCVR IgG1; FIG. 11J, CDR grafted 4E2 LCVR Kappa; FIG. 11K, CDR grafted3B3 HCVR IgG1; FIG. 11L, CDR grafted 3B3 LCVR Kappa; FIG. 11M, CDRgrafted 2H4 HCVR IgG1; FIG. 11N, CDR grafted 2H4 LCVR Kappa; FIG. 11O,CDR grafted 1F6 HCVR IgG1; FIG. 11P, veneered 20C2 HCVR VenA IgG1; FIG.11Q, veneered 20C2 HCVR VenB IgG1; FIG. 11R, veneered 20C2 HCVR VenBIgG2 m4; FIG. 11S, veneered 20C2 LCVR Kappa; FIG. 11T, veneered 26D6HCVR Ven1 Ig; FIG. 11U, veneered 26D6 HCVR Ven1 IgG1; FIG. 11V, 26D6HCVR Ven2 IgG1; FIG. 11W, veneered 26D6 HCVR Ven3; FIG. 11X, veneered26D6 LCVR Ven1 Kappa; and FIG. 11Y, veneered 26D6 LCVR Ven2 Kappa.Underlining indicates variable region sequences and amino acidscorresponding to the CDRs are double-underlined. The remaining aminoacid sequences are constant region sequences.

FIG. 12 shows a comparison of the amino acid sequence of human antibodyconstant regions and the sequence of IgG2 m4. The asterisk indicates aglycosylation site at Asn297. Regions of FcRn binding are indicated.Sequences in which IgG2 m4 is different from IgG2 are underlined.

FIG. 13 shows the annotated amino acid sequence for heavy (FIG. 13A) andlight (FIG. 13B) chains of 20C2 humanized antibody in Fab phage-displayvector pFab3d.

FIG. 14 depicts the design and primers employed in preparing two LC-CDR3libraries, namely LC3-1 and LC3-2, for generating an affinity matured20C2 light chain CDR3. Restriction endonuclease recognition sites usedfor cloning are indicated in italic. Uppercase indicates nucleic acidsencoding antibody variable region sequences. Nucleic acids encoding CDRsare underlined.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibodies, which differentially recognize multi-dimensionalconformations of Aβ-derived diffusible ligands (i.e., ADDLs), have nowbeen generated. Advantageously, the instant monoclonal antibodies candistinguish between Alzheimer's Disease and control human brainextracts, and identify endogenous oligomers in Alzheimer's Disease brainslices and in cultured hippocampal cells. Further, the instantantibodies neutralize endogenous and synthetic ADDLs in solution.So-called “synthetic” ADDLs are produced in vitro by mixing purifiedamyloid β1-42 under conditions that generate ADDLs. See U.S. Pat. No.6,218,506. Particular antibodies disclosed herein exhibit a high degreeof selectivity for 3-24mers, with minimal detection of monomer Aβpeptides. Further, recognition of ADDLs by selected antibodies of theinvention is not blocked by short peptides that encompass the linearsequence of Aβ1-42 or Aβ1-40. However, binding is blocked by Aβ1-28,indicating an epitope based on a conformationally unique structure alsofound in Aβ1-28. Delineation of epitopes of the instant antibodiesindicated that these antibodies recognize similar core linear sequenceswith similar affinity and specificity characteristics as measured byELISA. Moreover, the instant antibodies differentially block the abilityof ADDL-containing preparations to bind primary cultures of rathippocampal neurons and immortalized neuroblastoma cell lines, and alsoblock ADDL assembly. This finding demonstrates that these antibodiespossess a differential ability to recognize a multi-dimensionalconformation of ADDLs despite similar linear sequence recognition andaffinities. Since ADDLs are known to associate with a subset of neuronsand disrupt normal neuronal function, one use of this current inventionis the development and/or identification of antibodies that prevent thebinding of ADDLs to neurons. Such antibodies would be useful in thetreatment of ADDL related diseases including Alzheimer's Disease. Arefinement of this use would be to specifically use humanized and/oraffinity-matured versions of these antibodies for the prevention of ADDLbinding to neurons and assembly of ADDLs.

Accordingly, the present invention is an isolated antibody thatdifferentially recognizes one or more multi-dimensional conformations ofADDLs. An antibody of the instant invention is said to be isolated whenit is present in the substantial absence of other biologicalmacromolecules of the same type. Thus, an “isolated antibody” refers toan antibody which is substantially free of other antibodies; however,the molecule may include some additional agents or moieties which do notdeleteriously affect the basic characteristics of the antibody (e.g.,binding specificity, neutralizing activity, etc.).

Antibodies which are capable of specifically binding one or moremulti-dimensional conformations of ADDLs, bind particular ADDLs derivedfrom the oligomerization of Aβ1-42, but do not cross-react with other Aβpeptides, namely Aβ1-12, Aβ1-28, Aβ1-40, and Aβ12-28 as determined bywestern blot analyses as disclosed herein; and preferentially bind ADDLsin solution (see, e.g., Example 21). Specific binding between twoentities generally refers to an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹,or 10¹⁰ M⁻¹. Affinities greater than 10⁸ M⁻¹ are desired to achievespecific binding.

In particular embodiments, an antibody that is capable of specificallybinding a multi-dimensional conformation of one or more ADDLs is alsoraised against (i.e., an animal is immunized with) multi-dimensionalconformations of ADDLs. In other embodiments, an antibody that iscapable of specifically binding a multi-dimensional conformation of oneor more ADDLs is raised against a low n-mer-forming peptide such asAβ1-42-[Nle35-Dpro37].

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond or a site on a molecule against which an antibody will beproduced and/or to which an antibody will bind. For example, an epitopecan be recognized by an antibody defining the epitope.

A linear epitope is an epitope wherein an amino acid primary sequencecomprises the epitope recognized. A linear epitope typically includes atleast 3, and more usually, at least 5, for example, about 8 to about 10amino acids in a unique sequence.

A conformational epitope, in contrast to a linear epitope, is an epitopewherein the primary sequence of the amino acids comprising the epitopeis not the sole defining component of the epitope recognized (e.g., anepitope wherein the primary sequence of amino acids is not necessarilyrecognized by the antibody defining the epitope). Typically aconformational epitope encompasses an increased number of amino acidsrelative to a linear epitope. With regard to recognition ofconformational epitopes, the antibody recognizes a three-dimensionalstructure of the peptide or protein. For example, when a proteinmolecule folds to form a three-dimensional structure, certain aminoacids and/or the polypeptide backbone forming the conformational epitopebecome juxtaposed enabling the antibody to recognize the epitope.Methods of determining conformation of epitopes include but are notlimited to, for example, x-ray crystallography, two-dimensional nuclearmagnetic resonance spectroscopy and site-directed spin labeling andelectron paramagnetic resonance spectroscopy. See, for example, EpitopeMapping Protocols in Methods in Molecular Biology (1996) Vol. 66, Morris(Ed.).

Aβ-derived diffusible ligands or ADDLs refer to soluble oligomers ofamyloid β1-42 which are desirably composed of aggregates of less thaneight or nine amyloid β1-42 peptides and are found associated withAlzheimer's Disease. This is in contrast to high molecular weightaggregation intermediates, which form stings of micelles leading tofibril formation.

As exemplified herein, the instant antibody binds or recognizes at leastone multi-dimensional conformation of an ADDL (see, e.g., FIG. 3). Inparticular embodiments, the instant antibody binds at least two, atleast three, or at least four multi-dimensional conformations of anADDL. Multi-dimensional conformations of ADDLs are intended to encompassdimers, trimers, tetramers pentamers, hexamers, heptamers, octamers,nonamers, decamers, etc as defined by analysis via SDS-PAGE. Becausetrimer, tetramer, etc. designations can vary with the assay methodemployed (see, e.g., Bitan, et al. (2005) Amyloid 12:88-95) thedefinition of trimer, tetramer, and the like, as used herein, isaccording to SDS-PAGE analysis. To illustrate the differentially bindingcapabilities of the instant antibodies, it has been found that certainantibodies will recognize one multi-dimensional conformation, forexample, tetramers of ADDLs (e.g., antibody 2D6 or 4E2), while otherantibodies recognize several multi-dimensional conformations, forexample, trimers and tetramers of ADDLs (e.g., antibody 2A10, 2B4, 5F10,or 20C2). As such, the antibodies of the instant invention haveoligomer-specific characteristics. In particular embodiments, amulti-dimensional conformation of an ADDL is associated with a specificpolypeptide structure which results in a conformational epitope that isrecognized by an antibody of the present invention. In otherembodiments, an antibody of the invention specifically binds amulti-dimensional conformation ADDL having a size range of approximatelya trimer or tetramer, which have molecular weights in excess of >50 kDa.

In certain embodiments, in addition to binding to a multi-dimensionalconformation, the instant antibody binds to a selected linear epitope ofamyloid β1-42. A linear epitope of an ADDLs is intended as a four, five,six or more amino acid residue peptide located in the N-terminal 10, 11,12, 15 or 20 amino acid residues of amyloid β1-42. In particularembodiments, an antibody of the invention specifically binds to a linearepitope within residues 1-10, 1-8, 3-10, or 3-8 of amyloid β1-42.Exemplary linear epitopes of amyloid β 1-42 include, but are not limitedto, amino acid residues EFRHDS (SEQ ID NO:177); DAEFRHDS (SEQ IDNO:178), and EFRHDSGY (SEQ ID NO:179).

While antibodies of the instant invention may have similar linearepitopes, such linear epitopes are not wholly indicative of the bindingcharacteristics of the instant antibodies (i.e., ability to block ADDLbinding to neurons, prevent tau phosphorylation and inhibit ADDLassembly) because, as is well known to the skilled artisan, the linearepitope may only correspond to a portion of the antigen's epitope (see,e.g., Breitling and Dübel (1999) In: Recombinant Antibodies, John Wiley& Sons, Inc., NY, pg. 115). For example, 20C2 was found to bindassemblies of charge-inverted, truncated Aβ7-42 peptide, which lack thelinear epitope for 20C2 (i.e., amino acid residues 3-8) and contain avery different sequence corresponding to residues 7-16 of Aβ. Therefore20C2 binds to conformational epitopes that depend upon elements fromwithin residues 17-42 of Aβ, but only when in a multidimensionalconformation. The antibodies of the instant invention can bedistinguished from those of the art as being capable of differentiallyrecognizing multi-dimensional ADDLs and accordingly differentiallyblocking ADDL binding to neurons, differentially preventing tauphosphorylation and differentially inhibiting ADDL assembly.

An antibody, as used in accordance with the instant invention includes,but is not be limited to, polyclonal or monoclonal antibodies, andchimeric, human (e.g. isolated from B cells), humanized, neutralizing,bispecific or single chain antibodies thereof. In one embodiment, anantibody of the instant invention is monoclonal. For the production ofantibodies, various hosts including goats, rabbits, chickens, rats,mice, humans, and others, can be immunized by injection with syntheticor natural ADDLs. Methods for producing antibodies are well-known in theart. See, e.g., Kohler and Milstein ((1975) Nature 256:495-497) andHarlow and Lane (Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York (1988)).

Depending on the host species, various adjuvants can be used to increasethe immunological response. Adjuvants used in accordance with theinstant invention desirably augment the intrinsic response to ADDLswithout causing conformational changes in the immunogen that affect thequalitative form of the response. Particularly suitable adjuvantsinclude 3 De-O-acylated monophosphoryl lipid A (MPL™; RIBI ImmunoChemResearch Inc., Hamilton, Mont.; see GB 2220211) and oil-in-wateremulsions, such as squalene or peanut oil, optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute, etal. (1997) N. Engl. J. Med. 336:86-91), muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(E-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP)), or other bacterial cell wall components.Specific examples of oil-in-water emulsions include MF59 (WO 90/14837),containing 5% Squalene, 0.5% TWEEN™ 80, and 0.5% SPAN 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.); SAF containing 10% Squalene, 0.4% TWEEN™80, 5% PLURONIC®-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion; and RIBI™ adjuvant system (RAS) (RibiImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% TWEEN™ 80, andone or more bacterial cell wall components such as monophosphoryllipidA, trehalose dimycolate (TDM), and cell wall skeleton (CWS).

Another class of adjuvants is saponin adjuvants, such as STIMULON™(QS-21, Aquila, Framingham, Mass.) or particles generated therefrom suchas ISCOMs (immunostimulating complexes) and ISCOMATRIX® (CSL Ltd.,Parkville, Australia). Other suitable adjuvants include CompleteFreund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), mineralgels such as aluminum hydroxide, and surface-active substances such aslysolecithin, PLURONIC® polyols, polyanions, peptides, CpG (WO98/40100), keyhole limpet hemocyanin, dinitrophenol, and cytokines suchas interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulatingfactor (M-CSF), and tumor necrosis factor (TNF). Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum areparticularly suitable.

An antibody to a multi-dimensional conformation ADDL is generated byimmunizing an animal with ADDLs. Generally, ADDLs can be generatedsynthetically or by recombinant fragment expression and purification.Synthetic ADDLs can be prepared as disclosed herein or in accordancewith the methods disclosed in U.S. Pat. No. 6,218,506 or in co-pendingapplications U.S. Ser. No. 60/621,776, 60/652,538, 60/695,526 and60/695,528. Further, ADDLs can be fused with another protein such askeyhole limpet hemocyanin to generate an antibody against the chimericmolecule. The ADDLs can be conformationally constrained to form anepitope useful as described herein and furthermore can be associatedwith a surface for example, physically attached or chemically bonded toa surface in such a manner so as to allow for the production of aconformation which is recognized by the antibodies of the presentinvention.

Monoclonal antibodies to multi-dimensional conformations of ADDLs can beprepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Kohler, et al.(1975) Nature 256:495-497; Kozbor, et al. (1985) J. Immunol. Methods81:31-42; Cote, et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole,et al. (1984) Mol. Cell. Biol. 62:109-120). Exemplary monoclonalantibodies include murine antibodies designated 2A10, 4C2, 2D6, 4E2,20C2, 2B4, 5F10, 2H4, 2E12, 1F6, 1F4, 3B3, 5G12, 6B7, 6B11, 11B4, 11B5,14A11, 15G6, 17G4, 20C2, 3B7, 1E3, 1A9, 1G3, 1A7 and 1E5.

In addition, humanized and chimeric antibodies can be produced bysplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity(see Morrison, et al. (1984) Proc. Natl. Acad. Sci. 81, 6851-6855;Neuberger, et al. (1984) Nature 312:604-608; Takeda, et al. (1985)Nature 314:452-454; Queen, et al. (1989) Proc. Natl. Acad. Sci. USA86:10029-10033; WO 90/07861). For example, a mouse antibody is expressedas the Fv or Fab fragment in a phage selection vector. The gene for thelight chain (and in a parallel experiment, the gene for the heavy chain)is exchanged for a library of human antibody genes. Phage antibodies,which still bind the antigen, are then identified. This method, commonlyknown as chain shuffling, provided humanized antibodies that should bindthe same epitope as the mouse antibody from which it descends (Jespers,et al. (1994) Biotechnology NY 12:899-903). As an alternative, chainshuffling can be performed at the protein level (see, Figini, et al.(1994) J. Mol. Biol. 239:68-78).

Human antibodies can also be obtained using phage-display methods. See,e.g., WO 91/17271 and WO 92/01047. In these methods, libraries of phageare produced in which members display different antibodies on theirouter surfaces. Antibodies are usually displayed as Fv or Fab fragments.Phage displaying antibodies with a desired specificity are selected byaffinity enrichment to ADDLs. Human antibodies against ADDLs can also beproduced from non-human transgenic mammals having transgenes encoding atleast a segment of the human immunoglobulin locus and an inactivatedendogenous immunoglobulin locus. See, e.g., WO 93/12227 and WO 91/10741,each incorporated herein by reference. Human antibodies can be selectedby competitive binding experiments, or otherwise, to have the sameepitope specificity as a particular mouse antibody. Such antibodies areparticularly likely to share the useful functional properties of themouse antibodies. Human polyclonal antibodies can also be provided inthe form of serum from humans immunized with an immunogenic agent.Optionally, such polyclonal antibodies can be concentrated by affinitypurification using ADDLs as an affinity reagent.

Humanized antibodies can also be produced by veneering or resurfacing ofmurine antibodies. Veneering involves replacing only the surface fixedregion amino acids in the mouse heavy and light variable regions withthose of a homologous human antibody sequence. Replacing mouse surfaceamino acids with human residues in the same position from a homologoushuman sequence has been shown to reduce the immunogenicity of the mouseantibody while preserving its ligand binding. The replacement ofexterior residues generally has little, or no, effect on the interiordomains, or on the interdomain contacts. (See, e.g., U.S. Pat. No.6,797,492).

Human or humanized antibodies can be designed to have IgG, IgD, IgA, IgMor IgE constant regions, and any isotype, including IgG1, IgG2, IgG3 andIgG4. In particular embodiments, an antibody of the invention is IgG orIgM, or a combination thereof. A particular combination embraces aconstant region formed by selective incorporation of human IgG4sequences into a standard human IgG2 constant region. An exemplarymutant IgG2 Fc is IgG2 m4, set forth herein as SEQ ID NO:254. Antibodiescan be expressed as tetramers containing two light and two heavy chains,as separate heavy chains and light chains or as single chain antibodiesin which heavy and light chain variable domains are linked through aspacer. Techniques for the production of single chain antibodies arewell-known in the art.

Exemplary humanized antibodies produced by CDR grafting and veneeringare disclosed herein for antibodies designated 4E2, 26D6, 20C2, 3B3,2H4, and 1F6. Amino acid sequences for IgG1 and IgG2M4 heavy chainvariable regions, as well as kappa light chain variable regions forhumanized 4E2, 26D6, 20C2, 3B3, 2H4, and 1F6 generated by CDR graftingand veneering are presented in FIGS. 11A to 11Y and set forth herein asSEQ ID NOs:152 to 176.

Diabodies are also contemplated. A diabody refers to an engineeredantibody construct prepared by isolating the binding domains (both heavyand light chain) of a binding antibody, and supplying a linking moietywhich joins or operably links the heavy and light chains on the samepolypeptide chain thereby preserving the binding function (see, Holligeret al. (1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak (1994)Structure 2:1121-1123). This forms, in essence, a radically abbreviatedantibody, having only the variable domain necessary for binding theantigen. By using a linker that is too short to allow pairing betweenthe two domains on the same chain, the domains are forced to pair withthe complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or diabodies,are bivalent and bispecific. The skilled artisan will appreciate thatany method to generate diabodies can be used. Suitable methods aredescribed by Holliger, et al. (1993) supra, Poljak (1994) supra, Zhu, etal. (1996) Biotechnology 14:192-196, and U.S. Pat. No. 6,492,123,incorporated herein by reference.

Fragments of an isolated antibody of the invention are also expresslyencompassed by the instant invention. Fragments are intended to includeFab fragments, F(ab′)₂ fragments, F(ab′) fragments, bispecific scFvfragments, Fd fragments and fragments produced by a Fab expressionlibrary, as well as peptide aptamers. For example, F(ab′)₂ fragments areproduced by pepsin digestion of the antibody molecule of the invention,whereas Fab fragments are generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (see Huse, et al. (1989) Science254:1275-1281). In particular embodiments, antibody fragments of thepresent invention are fragments of neutralizing antibodies which retainthe variable region binding site thereof. Exemplary are F(ab′)₂fragments, F(ab′) fragments, and Fab fragments. See generallyImmunology: Basic Processes (1985) 2^(nd) edition, J. Bellanti (Ed.) pp.95-97.

Peptide aptamers which differentially recognize multi-dimensionalconformations of ADDLs can be rationally designed or screened for in alibrary of aptamers (e.g., provided by Aptanomics SA, Lyon, France). Ingeneral, peptide aptamers are synthetic recognition molecules whosedesign is based on the structure of antibodies. Peptide aptamers consistof a variable peptide loop attached at both ends to a protein scaffold.This double structural constraint greatly increases the binding affinityof the peptide aptamer to levels comparable to that of an antibody(nanomolar range).

Exemplary nucleic acid sequences encoding heavy and light chain variableregions for use in producing antibody and antibody fragments of theinstant invention are disclosed herein in FIGS. 6 and 10 (i.e., SEQ IDNOs:1-24 and SEQ ID NOs:132-151). As will be appreciated by the skilledartisan, the heavy chain variable regions disclosed herein can be usedin combination with any one of the light chain variable regionsdisclosed herein to generate antibodies with modified affinities,dissociate constants, epitopes and the like. For example, combining thelight chain variable region of 2H4 (encoded by SEQ ID NO:12) with theheavy chain variable region of 2A10 (encoded by SEQ ID NO:13) mayprovide for recognition of a larger linear epitope.

Exemplary heavy and light chain CDRs for use in producing an antibody orantibody fragment of the instant invention are disclosed in FIGS. 7A-7Fand have amino acid sequences set forth in SEQ ID NOs:25, 26, and 28(heavy chain CDR1); SEQ ID NOs: 29, 30, 31, 33, 34, 35, and 36 (heavychain CDR2); SEQ ID NOs:38, 39, 40, 41, 43, 44, 45, 46, 47 and 48 (heavychain CDR3); SEQ ID NOs:49, 50, 51 and 53 (light chain CDR1); SEQ IDNOs:54, 55, 56, and 58 (light chain CDR2); and SEQ ID NOs:59, 60, 61,62, 63, 64, and 66 (light chain CDR3). Particular embodiments of theheavy and light chains of the antibody or antibody fragments of theinstant invention are as follows. A heavy chain CDR1 having an aminoacid sequence of Ser-Phe-Gly-Met-His (SEQ ID NO:28) orThr-Ser-Gly-Met-Gly-Val-Xaa (SEQ ID NO:27), wherein Xaa is an amino acidwith no side chain or a small side chain (e.g., Ser, Gly, or Ala). Aheavy chain CDR2 having an amino acid sequence ofHis-Ile-Xaa₁-Trp-Asp-Asp-Asp-Lys-Xaa₂-Tyr-Asn-Pro-Ser-Leu-Lys-Ser (SEQID NO:32), wherein Xaa₁ is an amino acid with an aromatic side chaingroup (e.g., Phe, Tyr or Trp) and Xaa₂ is Ser, Arg or Tyr; or a heavychain CDR2 having an amino acid sequence ofTyr-Ile-Xaa₁-Xaa₂-Xaa₃-Ser-Xaa₄-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Arg(SEQ ID NO:37), wherein Xaa₁ and Xaa₂ are amino acids with a polar sidechain group (e.g., Arg, Ser, Gly, Thr, Cys, Tyr, Asn, Gln, Lys, or His);Xaa₃ is Gly or Val; and Xaa₄ is an amino acid with a polar and unchargedside group (e.g., Gly, Ser, Thr, Cys, Tyr, Asn, or Gln). A heavy chainCDR3 having an amino acid sequence ofArg-Ser-Ile-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Pro-Glu-Asp-Tyr-Phe-Xaa₅-Tyr (SEQ IDNO:42), wherein Xaa₁ is an amino acid with a polar and uncharged sidegroup (e.g., Gly, Ser, Thr, Cys, Tyr, Asn, or Gln); Xaa₂ is an aminoacid with hyroxyl side chain group (e.g., Ser or Thr); Xaa₃ and Xaa₄ areamino acids with an aliphatic side chain group (e.g., Ala, Val, Leu,Ile, or Pro); and Xaa₅ is Asp or Ala. A light chain CDR1 having an aminoacid sequence ofArg-Ser-Ser-Gln-Ser-Xaa₁-Xaa₂-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Xaa₃ (SEQID NO:52), wherein Xaa₁ and Xaa₂ are amino acids with an aliphatic sidechain group (e.g., Ala, Val, Leu, Ile, or Pro) and Xaa₃ is an amino acidwith a charged side chain group (e.g., Asp, Glu, Arg, His, or Lys). Alight chain CDR2 having an amino acid sequence ofLys-Xaa₁-Ser-Asn-Arg-Phe-Xaa₂ (SEQ ID NO:57), wherein Xaa₁ is an aminoacid with an aliphatic side chain group (e.g., Ala, Val, Leu, Ile, orPro) and Xaa₂ is Ser or Phe. A light chain CDR3 having an amino acidsequence of Xaa₁-Gln-Xaa₂-Xaa₃-Xaa₄-Val-Pro-Xaa₅-Thr (SEQ ID NO:65),wherein Xaa₁ is Ser or Phe; Xaa₂ is an amino acid with no side chain(e.g., gly) or hyroxyl side chain group (e.g., Ser or Thr); Xaa₃ is anamino acid with a hyroxyl side chain group (e.g., Ser or Thr); Xaa₄ isHis, Tyr or Leu; and Xaa₅ is an amino acid with an aliphatic side chaingroup (e.g., Ala, Val, Leu, Ile, or Pro). As will be appreciated by theskilled artisan, one or more of the CDRs within the heavy and lightchain variable regions of an antibody can be replaced with one or moreCDRs from another antibody to generate a wholly new antibody or antibodyfragment. For example, replacing CDR3 of the heavy chain of 5F10 withthe CDR3 of the heavy chain from 4E2 (SEQ ID NO:41) may enhance thatability of 5F10 to block binding of ADDLs to neuronal cells.

Antibodies with particular characteristics are contemplated. In oneembodiment, an antibody which binds the 3-8 amino acid epitope of Aβ1-42has a heavy chain CDR1 amino acid sequence ofThr-Ser-Gly-Met-Gly-Val-Xaa (SEQ ID NO:27), wherein Xaa is an amino acidwith no side chain or a small side chain (e.g., Ser, Gly, or Ala); or aheavy chain CDR2 amino acid sequence ofHis-Ile-Xaa₁-Trp-Asp-Asp-Asp-Lys-Xaa₂-Tyr-Asn-Pro-Ser-Leu-Lys-Ser (SEQID NO:32), wherein Xaa₁ is an amino acid with an aromatic side chaingroup (e.g., Phe, Tyr or Trp) and Xaa₂ is Ser, Arg or Tyr. In anotherembodiment, an antibody with a moderate affinity for large (>50 kDa)ADDL aggregates over small (<30 kDa) aggregates (i.e. SEC Peak 1 andPeak 2, respectively), has a heavy chain CDR3 amino acid sequence ofArg-Ser-Ile-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Pro-Glu-Asp-Tyr-Phe-Xaa₅-Tyr (SEQ IDNO:42), wherein Xaa₁ is an amino acid with a polar and uncharged sidegroup (e.g., Gly, Ser, Thr, Cys, Tyr, Asn, or Gln), Xaa₂ is an aminoacid with hyroxyl side chain group (e.g., Ser or Thr), Xaa₃ and Xaa₄ areamino acids with an aliphatic side chain group (e.g., Ala, Val, Leu,Ile, or Pro), and Xaa₅ is Asp or Ala.

Antibodies or antibody fragments of the present invention can haveadditional moieties attached thereto. For example, a microsphere ormicroparticle can be attached to the antibody or antibody fragment, asdescribed in U.S. Pat. No. 4,493,825, the disclosure of which isincorporated herein by reference.

Moreover, antibody or antibody fragments of the invention can be mutatedand selected for increased antigen affinity, neutralizing activity(i.e., the ability to block binding of ADDLs to neuronal cells or theability to block ADDL assembly), or a modified dissociation constant.Mutator strains of E. coli (Low, et al. (1996) J. Mol. Biol.260:359-368), chain shuffling (Figini, et al. (1994) supra), and PCRmutagenesis are established methods for mutating nucleic acid moleculesencoding antibodies. By way of illustration, increased affinity can beselected for by contacting a large number of phage antibodies with a lowamount of biotinylated antigen so that the antibodies compete forbinding. In this case, the number of antigen molecules should exceed thenumber of phage antibodies, but the concentration of antigen should besomewhat below the dissociation constant. Thus, predominantly mutatedphage antibodies with increased affinity bind to the biotinylatedantigen, while the larger part of the weaker affinity phage antibodiesremains unbound. Streptavidin can then assist in the enrichment of thehigher affinity, mutated phage antibodies from the mixture (Schier, etal. (1996) J. Mol. Biol. 255:28-43). Exemplary affinity-maturated lightchain CDR3 amino acid sequences are disclosed herein (see Tables 11 and12), with particular embodiments embracing a light chain CDR3 amino acidsequence of Xaa₁-Gln-Xaa₂-Thr-Arg-Val-Pro-Leu-Thr (SEQ ID NO:316),wherein Xaa₁ is Phe or Leu, and Xaa₁ is Ala or Thr.

For some therapeutic applications it may be desirable to reduce thedissociation of the antibody from the antigen. To achieve this, thephage antibodies are bound to biotinylated antigen and an excess ofunbiotinylated antigen is added. After a period of time, predominantlythe phage antibodies with the lower dissociation constant can beharvested with streptavidin (Hawkins, et al. (1992) J. Mol. Biol.226:889-96).

Various immunoassays including those disclosed herein can be used forscreening to identify antibodies, or fragments thereof, having thedesired specificity for multi-dimensional conformations of ADDLs.Numerous protocols for competitive binding (e.g, ELISA), latexagglutination assays, immunoradiometric assays, kinetics (e.g., BIACORE™analysis) using either polyclonal or monoclonal antibodies, or fragmentsthereof, are well-known in the art. Such immunoassays typically involvethe measurement of complex formation between a specific antibody and itscognate antigen. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes issuitable, but a competitive binding assay can also be employed. Suchassays can also be used in the detection of multi-dimensionalconformations of ADDLs in a sample.

An antibody or antibody fragment can also be subjected to otherbiological activity assays, e.g., displacement of ADDL binding toneurons or cultured hippocampal cells or blockade of ADDL assembly, inorder to evaluate neutralizing or pharmacological activity and potentialefficacy as a prophylactic or therapeutic agent. Such assays aredescribed herein and are well-known in the art.

Antibodies and fragments of antibodies can be produced and maintained ashydridomas or alternatively recombinantly produced in anywell-established expression system including, but not limited to, E.coli, yeast (e.g., Saccharomyces spp. and Pichia spp.), baculovirus,mammalian cells (e.g., myeloma, CHO, COS), plants, or transgenic animals(Breitling and Dübel (1999) In: Recombinant Antibodies, John Wiley &Sons, Inc., NY, pp. 119-132). Exemplary nucleic acid sequences of IgG1and IgG2 m4 heavy chain variable regions, as well as kappa light chainvariable regions for humanized 4E2, 26D6, 20C2, 3B3, 2H4, and 1F6generated by CDR grafting and veneering are presented in FIGS. 10A to10S and set forth herein as SEQ ID NOs:132 to 151. For antibodies andfragments of antibodies can be isolated using any appropriate methodsincluding, but not limited to, affinity chromatography,immunoglobulins-binding molecules (e.g., proteins A, L, G or H), tagsoperatively linked to the antibody or antibody fragment (e.g., His-tag,FLAG®-tag, Strep tag, c-myc tag) and the like. See, Breitling and Dübel(1999) supra.

Antibodies and antibody fragments of the instant invention have avariety of uses including, diagnosis of diseases associated withaccumulation of ADDLs, blocking or inhibiting binding of ADDLs toneuronal cells, blocking ADDL assembly, prophylactically ortherapeutically treating a disease associated with ADDLs, identifyingtherapeutic agents that prevent binding of ADDLs to neurons, andpreventing the phosphorylation of tau protein at Ser202/Thr205.

Antibody and antibody fragments of the instant invention are also usefulin a method for blocking or inhibiting binding of ADDLs to neuronalcells. This method of the invention is carried out by contacting aneuron, in vitro or in vivo, with an antibody or antibody fragment ofthe present invention so that binding of ADDLs to the neuron is blocked.In particular embodiments, an antibody or antibody fragment of theinstant invention achieves at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 97% decrease in the binding of ADDLs as compared tobinding of ADDLs in the absence of the antibody or antibody fragment.The degree to which an antibody can block the binding of ADDLs to aneuron can be determined in accordance with the methods disclosedherein, i.e., immunocytochemistry or cell-based alkaline phosphataseassay or any other suitable assay. Antibodies particularly useful fordecreasing binding of ADDLs to neuronal cells include the exemplary20C2, 3B3, 1F4, 1F6, 4E2, 2B4, 2D6, and 2H4 monoclonal antibodies.

Antibody and antibody fragments of the instant invention are furtheruseful in a method for blocking or inhibiting assembly of ADDLs. Thismethod involves contacting a sample containing amyloid β 1-42 peptideswith an antibody or antibody fragment of the instant invention so thatADDL assembly is inhibited. The degree to which an antibody can blockthe assembly of ADDLs can be determined in accordance with the methodsdisclosed herein, i.e., FRET or fluorescence polarization or any othersuitable assay. Antibodies particularly useful for blocking the assemblyof ADDLs include the exemplary 1F4, 20C2, 4C2, 1F6, 2B4, 5F10, 2A10, and2D6 antibodies.

Antibodies disclosed herein are also useful in methods for preventingthe phosphorylation of tau protein at Ser202/Thr205. This methodinvolves contacting a sample containing tau protein with an antibody orantibody fragment of the instant invention so that binding of ADDLs toneurons is blocked thereby preventing phosphorylation of tau protein.The degree to which an antibody can prevent the phosphorylation of tauprotein at Ser202/Thr205 can be determined in accordance with themethods disclosed herein or any other suitable assay.

Blocking or decreasing binding of ADDLs to neurons, inhibiting assemblyof ADDLs, and preventing the phosphorylation of tau protein atSer202/Thr205 all find application in methods of prophylactically ortherapeutically treating a disease associated with the accumulation ofADDLs. Accordingly, the present invention also embraces the use of anantibody or antibody fragment of the instant invention to prevent ortreat a disease associated with the accumulation of ADDLs (e.g.Alzheimer's or similar memory-related disorders). Patients amenable totreatment include individuals at risk of disease but not exhibitingsymptoms, as well as patients presently exhibiting symptoms. In the caseof Alzheimer's Disease, virtually anyone is at risk of suffering fromAlzheimer's Disease if he or she lives long enough. Therefore, theantibody or antibody fragments of the present invention can beadministered prophylactically to the general population without the needfor any assessment of the risk of the subject patient. The presentmethods are especially useful for individuals who have a known geneticrisk of Alzheimer's Disease. Such individuals include those havingrelatives who have been diagnosed with the disease, and those whose riskis determined by analysis of genetic or biochemical markers. Geneticmarkers of risk for Alzheimer's Disease include mutations in the APPgene, particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively. Othermarkers of risk are mutations in the presenilin genes, PS1 and PS2, andApoE4, family history of Alzheimer's Disease, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sDisease can be recognized from characteristic dementia, as well as thepresence of risk factors described above. In addition, a number ofdiagnostic tests are available for identifying individuals who haveAlzheimer's Disease. These include measurement of CSF tau and Aβ1-42levels. Individuals suffering from Alzheimer's Disease can also bediagnosed by ADRDA criteria or the method disclosed herein.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30 years of age). Usually, however, it is not necessary to begintreatment until a patient reaches 40, 50, 60 or 70 years of age.Treatment typically entails multiple dosages over a period of time.Treatment can be monitored by assaying for the presence of ADDLs overtime.

In therapeutic applications, a pharmaceutical composition or medicamentcontaining an antibody or antibody fragment of the invention isadministered to a patient suspected of, or already suffering from such adisease associated with the accumulation of ADDLs in an amountsufficient to cure, or at least partially arrest, the symptoms of thedisease (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. In prophylactic applications, a pharmaceutical compositionor medicament containing an antibody or antibody fragment of theinvention is administered to a patient susceptible to, or otherwise atrisk of, a disease associated with the accumulation of ADDLs in anamount sufficient to achieve passive immunity in the patient therebyeliminating or reducing the risk, lessening the severity, or delayingthe outset of the disease, including biochemical, histologic and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease. Insome methods, administration of agent reduces or eliminates myocognitiveimpairment in patients that have not yet developed characteristicAlzheimer's pathology. In particular embodiments, an effective amount ofan antibody or antibody fragment of the invention is an amount whichachieves at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or97% decrease in the binding of ADDLs to neurons in the patient ascompared to binding of ADDLs in the absence of treatment. As such,impairment of long-term potentiation/memory formation is decreased.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human but nonhuman mammals suchas dogs or transgenic mammals can also be treated.

Treatment dosages are generally titrated to optimize safety andefficacy. For passive immunization with an antibody or antibodyfragment, dosage ranges from about 0.0001 to 100 mg/kg, and more usually0.01 to 5 mg/kg, of the host body weight are suitable. For example,dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per every two weeks or once a month or once every 3to 6 months. In some methods, two or more antibodies of the inventionwith different binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Antibodies are usually administered on multipleoccasions, wherein intervals between single dosages can be weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to ADDLs in the patient. In somemethods, dosage is adjusted to achieve a plasma antibody concentrationof 1-1000 μg/mL and in some methods 25-300 μg/mL. Alternatively, theantibody or antibody fragment can be administered as a sustained-releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human and humanized antibodies have longerhalf-lives than chimeric antibodies and nonhuman antibodies. Asindicated above, dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Antibody and antibody fragments of the instant invention can beadministered as a component of a pharmaceutical composition ormedicament. Pharmaceutical compositions or medicaments generally containthe active therapeutic agent and a variety of other pharmaceuticallyacceptable components. See Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams &Wilkins: Philadelphia, Pa., 2000. The preferred form depends on theintended mode of administration and therapeutic application.Pharmaceutical compositions can contain, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers or diluents,which are defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. Diluents are selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution.

Pharmaceutical compositions can also contain large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such aslatex-functionalized SEPHAROSE™, agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

Administration of a pharmaceutical composition or medicament of theinvention can be carried out via a variety of routes including, but notlimited to, oral, topical, pulmonary, rectal, subcutaneous, intradermal,intranasal, intracranial, intramuscular, intraocular, or intra-articularinjection, and the like. The most typical route of administration isintravenous followed by subcutaneous, although other routes can beequally effective. Intramuscular injection can also be performed in thearm or leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for example,intracranial injection. In some embodiments, an antibody or antibodyfragment is injected directly into the cranium. In other embodiments,antibody or antibody fragment is administered as a sustained-releasecomposition or device, such as a MEDIPAD™ device.

For parenteral administration, antibody or antibody fragments of theinvention can be administered as injectable dosages of a solution orsuspension of the substance in a physiologically acceptable diluent witha pharmaceutical carrier that can be a sterile liquid such as water,oils, saline, glycerol, or ethanol. Additionally, auxiliary substances,such as wetting or emulsifying agents, surfactants, pH bufferingsubstances and the like can be present in compositions. Other componentsof pharmaceutical compositions are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,and mineral oil. In general, glycols such as propylene glycol orpolyethylene glycol are suitable liquid carriers, particularly forinjectable solutions. Antibodies can be administered in the form of adepot injection or implant preparation which can be formulated in such amanner as to permit a sustained-release of the active ingredient Anexemplary composition contains an antibody at 5 mg/mL, formulated inaqueous buffer composed of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced delivery.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, or more desirably 1%-2%.

Oral formulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained-releaseformulations or powders and contain 10%-95% of active ingredient, ormore suitably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (see Glenn, et al. (1998) Nature391:851). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul, et al. (1995) Eur. J. Immunol. 25:3521-24;Cevc, et al. (1998) Biochem. Biophys. Acta 1368:201-15).

An antibody or antibody fragment of the invention can optionally beadministered in combination with other agents that are at least partlyeffective in treatment of amyloidogenic disease.

Antibody and antibody fragments of the instant invention also findapplication in the identification of therapeutic agents that prevent thebinding of ADDLs to neurons (e.g. a hippocampal cell) thereby preventingdownstream events attributed to ADDLs. Such an assay is carried out bycontacting a neuron with ADDLs in the presence of an agent and using anantibody of antibody fragment of the invention to determine binding ofthe ADDLs to the neuron in the presence of the agent. As will beappreciated by the skilled artisan, an agent that blocks binding ofADDLs to a neuron will decrease the amount of ADDLs bound to the neuronas compared to a neuron which has not been contacted with the agent; anamount which is detectable in an immunoassay employing an antibody orantibody fragment of the instant invention. Suitable immunoassays fordetecting neuronal-bound ADDLs are disclosed herein.

Agents which can be screened using the method provided herein encompassnumerous chemical classes, although typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 100 and less than about 2,500 daltons. Agents encompassfunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The agents often contain cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Agents canalso be found among biomolecules including peptides, antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Agents are obtained from awide variety of sources including libraries of natural or syntheticcompounds.

A variety of other reagents such as salts and neutral proteins can beincluded in the screening assays. Also, reagents that otherwise improvethe efficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, and the like can be used. The mixtureof components can be added in any order that provides for the requisitebinding.

Agents identified by the screening assay of the present invention willbe beneficial for the treatment of amyloidogenic diseases and/ortauopathies. In addition, it is contemplated that the experimentalsystems used to exemplify these concepts represent research tools forthe evaluation, identification and screening of novel drug targetsassociated with amyloid beta induction of tau phosphorylation.

The present invention also provides methods for detecting ADDLs anddiagnosing a disease associated with accumulation of ADDLs using anantibody or antibody fragment of the instant invention. A diseaseassociated with accumulation of ADDLs is intended to include any diseasewherein the accumulation of ADDLs results in physiological impairment oflong-term potentiation/memory formation. Diseases of this type include,but are not limited to, Alzheimer's Disease and similar memory-relateddisorders.

In accordance with these methods, a sample from a patient is contactedwith an antibody or antibody fragment of the invention and binding ofthe antibody or antibody fragment to the sample is indicative of thepresence of ADDLs in the sample. As used in the context of the presentinvention, a sample is intended to mean any bodily fluid or tissue whichis amenable to analysis using immunoassays. Suitable samples which canbe analyzed in accordance with the methods of the invention include, butare not limited to, biopsy samples and fluid samples of the brain from apatient (e.g., a mammal such as a human). For in vitro purposes (e.g.,in assays monitoring oligomer formation), a sample can be a neuronalcell line or tissue sample. For diagnostic purposes, it is contemplatedthat the sample can be from an individual suspected of having a diseaseassociated with accumulation of ADDLs or from an individual at risk ofhaving a disease associated with accumulation of ADDLs, e.g., anindividual with a family history which predisposes the individual to adisease associated with accumulation of ADDLs.

Detection of binding of the antibody or antibody fragment to ADDLs inthe sample can be carried out using any standard immunoassay (e.g., asdisclosed herein), or alternatively when the antibody fragment is, e.g.,a peptide aptamer, binding can be directly detected by, for example, adetectable marker protein (e.g., β-galactosidase, GFP or luciferase)fused to the aptamer. Subsequently, the presence or absence of theADDL-antibody complex is correlated with the presence or absence,respectively, of ADDLs in the sample and therefore the presence orabsence, respectively, of a disease associated with accumulation ofADDLs. It is contemplated that one or more antibodies or antibodyfragments of the present invention can be used in conjunction withcurrent non-invasive immuno-based imaging techniques to greatly enhancedetection and early diagnosis of a disease associated with accumulationof ADDLs.

To facilitate diagnosis the present invention also pertains to a kit forcontaining an antibody or antibody fragment of the instant invention.The kit includes a container holding one or more antibody or antibodyfragments which recognizes multi-dimensional conformation of ADDLs andinstructions for using the antibody for the purpose of binding to ADDLsto form an antibody-antigen complex and detecting the formation of theantibody-antigen complex such that the presence or absence of theantibody-antigen complex correlates with presence or absence of ADDLs inthe sample. Examples of containers include multiwell plates which allowsimultaneous detection of ADDLs in multiple samples.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 General Materials and Methods

ADDL Preparation. ADDLs in F12 medium (Biosource, Camarillo, Calif.)were prepared from Aβ1-42 in accordance with established methods(Lambert, et al. (2001) supra). Briefly, Aβ1-42 peptide (AmericanPeptide Co., Sunnyvale, Calif. or California Peptide Research, Inc.,Napa, Calif.) was weighed and placed in a glass vial capable of holdinga sufficient quantity of HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) toachieve a peptide concentration of 10 mg/mL. HFIP was added to the drypeptide, the vial was capped and gently swirl to mix, and thepeptide/HFIP solution was stored at room temperature for at least onehour. Aliquots (50 or 100 μL, 0.5 or 1.0 mg, respectively) of peptidesolution was dispensed into a series of 1.5 mL conical centrifuge tubes.The tubes were placed in a speedvac overnight to remove the HFIP. Tubescontaining the dried peptide film were capped and stored at −70° C. in asealed container with dessicant.

Prior to use, the Aβ1-42 peptide film was removed from −70° C. storageand allowed to warm to room temperature. Fresh DMSO (44 μL/mg of peptidefilm; 5 mM) was added and the peptide/DMSO mixture was incubated on avortex mixer at the lowest possible speed for ten minutes. F12 media (2mL/mg peptide) was dispensed into each tube of DMSO/peptide and the tubewas capped and mixed by inversion. The 100 μM preparation was stored at2-8° C. for eighteen to twenty four hours. The samples were centrifugedat 14,000×g for ten minutes at 2-8° C. The supernatant was transferredto a fresh tube and stored at 2-8° C. until used.

Biotinylated ADDL preparations (bADDLs) were prepared in the same manneras described above for ADDL preparations using 100% N-terminalbiotinylated amyloid beta peptide (American Peptide Company, Sunnyvale,Calif.).

ADDL Fibril Preparation. To room temperature ADDL peptide film was added2 mL of 10 mM hydrochloric acid per mg peptide. The solution was mixedon a vortex mixer at the lowest possible speed for five to ten minutesand the resulting preparation was stored at 37° C. for eighteen totwenty four hours before use.

Monomer Preparation. HFIP dry down preparations of amyloid beta (1-40)peptide (Aβ1-40) were prepared as outlined for Aβ(1-42) peptide. Thepeptide film was dissolved in 2 mL of 25 mM borate buffer (pH 8.5) permg of peptide, divided into aliquots, and frozen at −70° C. until used.

Human Fibril Preparation. Samples obtained from frozen human cortex werehomogenized in 20× cold F12 medium with protease inhibitors (COMPLETE®,Roche Diagnostics Corporation, Indianapolis, Ind.) for 1 minute. Thesample was then centrifuged at 10,000×g for 1 hour at 4° C. Afterwashing twice with F12, the pellet was resuspended in 2% SDS/F12 andincubated on ice for 30 minutes. The sample was subsequently centrifugedat 220,000×g for 1 hour at 4° C. The pellet was resuspended in cold F12and sonicated for 1 minute in 15-second bursts. Protein was determinedusing COOMASSIE PLUS™ kit (Pierce Biotechnology, Rockford, Ill.).

Immunization. The resulting soluble Aβ oligomers, referred to herein as“synthetic” ADDLs, were mixed 1:1 with complete Freund's adjuvant (firstand second vaccination) or incomplete Freund's adjuvant (all subsequentvaccinations) and injected subcutaneously (first two vaccinations) orintraperitoneally into three mice in a total volume of ˜1 mL/mouse. Eachinjection consisted of purified ADDLs equivalent to 194±25 μg totalprotein. Mice were injected approximately every three weeks. After sixinjections, one mouse died and its spleen was frozen. The spleen fromthe mouse with the highest titer serum was then fused with SP2/0 myelomacells in the presence of polyethylene glycol and plated out into six96-well plates. The cells were cultured at 37° C. with 5% CO₂ for tendays in 200 μL of HAT selection medium, which is composed of ISCOVmedium supplemented with 10% fetal bovine serum (FBS), 1 μg/mL HYBRIMAX®(azaserine-hypoxanthine; Sigma-Aldrich, St. Louis, Mo.), and 30%conditioned media collected from SP2/0 cell culture. The cultures werefed once with ISCOV medium supplemented with 10% FBS on day 10, and theculture supernatants were removed on day 14 to screen for positive wellsin ELISA. The positive cultures were further cloned by limitingdilutions with probability of 0.3 cells per well. The positive cloneswere confirmed in ELISA and further expanded.

Screening of supernates involved five assays: a dot blot and westernimmunoblot (Lambert, et al. (2001) supra), a native immunoblot usingsynthetic ADDLs, and a dot blot and western blot using endogenousfibrils obtained from human tissue. These assays tested the binding ofantibodies to ADDLs (the dot blot) and identified the oligomer(s) thathad the greatest affinity (western). All antibodies were tested in thedot blot using 5 pmole ADDLs (576 supernates in the first fusion and1920 supernates in the second). Those supernatants that tested positivewere then screened further using western blot at 10-20 pmole ADDLs. Thescreen was repeated to identify low positives or false positives. Tenwells supernatants expanded for the first mouse and forty-five wellswere expanded for the second mouse. The expanded cells were then frozenor subcloned.

Monoclonal antibody-containing ascites were produced in female balb/cmice using standard protocols (Current Protocol of Molecular Biology).Briefly, mice were primed by intraperitoneal injection of 0.5 mL ofpristane. One week after the priming, mice were injectedintraperitoneally with approximately 5×10⁶ hybridoma cells in 1 mLphosphate-buffered saline (PBS). Ascites were collected ten to fourteendays later. IgG purification was carried out by using BIO-RAD® AFFI-GEL®Protein A MAPS® II kit, according to manufacturer's protocol. For eachrun, 3 mL ascites were desalted by passage through a desalting columnand elution in 4 mL binding buffer. The sample was then applied to theProtein A column. After washing with 40 mL binding buffer, the columnwas eluted with elution buffer and the 5 mL fractions were collected.Samples were neutralized by addition of 60 μL of 10 N NaOH. To exchangethe buffer to PBS, the samples were applied to a second desalting columnand eluted with PBS.

Control Antibodies. Polyclonal antibodies M71/2 and M90/1 were obtainedfrom Bethyl Laboratories, Inc. (Montgomery, Tex.). Anti-Aβ monoclonalantibodies 6E10 (raised against residues 1-17) and 4G8 (raised againstresidues 17-24) were obtained from Signet Labs (Dedham, Mass.).Monoclonal antibody WO-2 is known in the art for its ability torecognize both 1-40 and 1-42 via western blot analysis (Ida, et al.(1996) J. Biol. Chem. 271: 22908-22914. Monoclonal antibody BAM-10(raised against Aβ1-40) was obtained from ABCAM® (Cambridge, Mass.).Monoclonal antibody 26D6 is well-known in the art for its ability torecognize amino acids 1-12 of Aβ sequence (Lu, et al. (2000) Nat. Med.6:397-404).

Immunoblot Analysis. Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) was performed using established methods(Lambert, et al. (2001) supra), except that 10-20% Tris-Tricine gels(BIO-RAD®, Hercules, Calif.) were used and the separation was performedat 120 V. Gels were transferred according to standard methods andsecondary antibody was routinely used at a 1:40,000 dilution.

For initial screening, 2.7 μg ADDLs, equivalent to ˜16-20 μmol/lane,were separated on two-dimensional (2D) 4-20% gels. Electrophoresis andtransfer were as above. Using the tracking dye as a guide, thenitrocellulose was placed into a Surf-blot apparatus (Idea Scientific,Minneapolis, Minn.) and 200 μL of hybridoma supernate mixed withblocking buffer, composed of 5% nonfat dry milk in tris-buffered salinewith TWEEN™ 20 (TBS-T; Lambert et al. (2001) supra), was added to eachof 20-21 wells. After incubation at room temperature for 1.5 hour withrocking, the supernatants were removed and the wells were washed with200 μL blocking buffer. The membrane was then removed from the Surf-blotapparatus and washed 3×15 minutes in TBS-T. The secondary antibody(anti-mouse, IgG conjugated-HRP, 1:40,000; Molecular Probes, Eugene,Oreg.) was then incubated with the membrane for 1 hour at roomtemperature. After washing (3×15 minutes), the oligomers were visualizedwith half-strength SUPERSIGNAL® (Pierce, Rockland, Ill.). The westernimmunoblot using human fibrils was performed in the same manner usingapproximately 64 μg of human fibrillar tissue in each 2D SDS-PAGEimmunoblot.

Native polyacrylamide gel electrophoresis was performed accordingestablished methods (Chromy, et al. (2003) Biochemistry 42:12749-12760)except that the separation was performed at 120 V.

Western Blot. Separated proteins were transferred to nitrocellulose.Blots were blocked with 5% non-fat dry milk or 1% bovine serum albumin(BSA) in TBS-T (TBS with 0.1% TWEEN™ 20) overnight, incubated withprimary antibody(ies) for 1.5 hours, washed, and incubated thehorseradish peroxidase (HRP)-conjugated secondary antibody (AmershamBiosciences Corp., Piscataway, N.J.) for 1 hour. After final washing,proteins were visualized with a West Femto chemiluminescence kit (PierceBiotechnology, Rockford, Ill.) and a KODAK® Image Station 440 CF or withfilm (HYPERFILM™, Amersham Biosciences Corp., Piscataway, N.J.).

Hippocampal Cultures. Cultures were prepared from E18 embryos accordingto standard methods (Brewer (1997) J. Neurosci. Methods 71:143-155;Stevens, et al. (1996) J. Neurosci. Res. 46:445-455). Viable cells werecounted and plated on coverslips coated with polylysine (200 μg/mL) atdensities from 1.5×10⁴-10⁶ cells/cm². The medium was changed by removinghalf of the medium and replacing it with supplemented NEUROBASAL™ media.

Primary Neurons. Primary hippocampal cultures were prepared from frozen,dissociated neonatal rat hippocampal cells (Cambrex, Corp., EastRutherford, N.J.) that were thawed and plated in 96-well COSTAR® platesat a concentration of 20,000 cells per well. The cells were maintainedin NEUROBASALT™ media without L-glutamine (GIBCO-BRL™, Gaithersburg,Md.) and supplemented with B27 (GIBCO-BRL™, Gaithersburg, Md.) for aperiod of two weeks and then used for binding studies.

B103 Cells. The B103 neuroblastoma cell line (Schubert and Behl (1993)Brain Res. 629:275-82) was grown in DMEM without phenol red(GIBCO-BRLT™, Gaithersburg, Md.), in the presence of 10% FBS (Hyclone,Logan, Utah) and 1% Pen-Strep (GIBCO-BRL™, Gaithersburg, Md.).Exponentially growing B103 cells were dissociated and plated in 96-wellCORNING® plates at a concentration of 5,000 cells/well. Twenty-fourhours after plating, the cells were used to assess ADDL and bADDLbinding as well as characterize commercial and novel anti-ADDLmonoclonal antibodies.

Dot Blot Analysis. Dot blots were performed according to Lambert, et al.((2001) supra) applying either ADDLs (5 pmole/dot) or fibrils to thenitrocellulose. For later dot blots, ADDLs were applied to drynitrocellulose in duplicate at various pmolar concentrations in 0.5 μLusing a template derived from the Surf-blot apparatus. Samples were thendried for 15 minutes, blocked with blocking buffer for 1 hour, andincubated for 1.5 hour with antibody plus or minus peptide, which hadbeen pre-incubated for at least 1 hour at room temperature. The solutionwas removed from the Surf-blot apparatus, the wells were washed withblocking buffer, and the membrane was removed from the apparatus. Thenitrocellulose was washed, treated with secondary antibody, andvisualized as indicated above.

Immunocytochemistry. Immunocytochemistry was performed according toestablished methods (Lambert, et al. (2001) supra), except the secondaryantibodies were conjugated to ALEXAFLUOR® 588 (Molecular Probes, Eugene,Oreg.). Antibodies and ADDLs were preincubated for 1 hour at roomtemperature, at a molar ratio of 1:4 antibody:ADDL before application tothe 21-day hippocampal cell culture. For endogenous ADDLs, human brainprotein (prepared as in Lambert, et al. (2001) supra) was incubated withcells for 1 hour before the cells were washed, fixed, and visualized asabove.

Lightly fixed frozen sections (4% paraformaldehyde at 4° C. for 30 hoursand cryoprotected in 40 μm sucrose) from Alzheimer's Disease and controlhippocampus were incubated with antibody (1:1000 in phosphate-bufferedsaline (PBS)) overnight at 4° C. After removal of antibody, sectionswere washed 3 times with PBS and incubated with secondary antibody atroom temperature. Binding was then visualized with DAB (SIGMAT™, St.Louis, Mo.). Sections were then counterstained with hematoxylin,mounted, and imaged on a NIKON® ECLIPSE® E600 light microscope with aSPOT™ INSIGHT™ digital video camera (v. 3.2).

Quantitative Immunocytochemistry. Cultured hippcampal cells wereincubated with 500 nM ADDLs for 1 hour at 37° C. ADDLs were removed bywashing and cells were fixed with 3.7% formaldehyde. Cells wereincubated with 0.1% TRITON™ X-100 in PBS-NGS (PBS with 10% normal goatserum) for 30 minutes, washed once, and incubated with the desiredprimary antibody(ies) (diluted in PBS-NGS) overnight at 4° C. Sampleswere washed and incubated with the appropriate secondary antibody(ies),e.g., ALEXAFLUOR® 488 or 594 anti-mouse and anti-rabbit IgGs (MolecularProbes, Inc., Eugene, Oreg.), for 2 hours at 37° C. Coverslips werewashed and mounted in PROLONG® anti-fade mounting medium (MolecularProbes, Inc., Eugene, Oreg.) and imaged using a LEICA® TCS SP2 confocalScanner DMRXE7 microscope.

ELISA. Polyclonal anti-ADDLs IgG (M90/1; Bethyl Laboratories, Inc.,Montgomery, Tex.) was plated at 0.25 mg/well on IMMULON™ 3 REMOVAWELL™strips (Dynatech Labs, Chantilly, Va.) for 2 hours at room temperatureand the wells blocked with 2% BSA in TBS. Samples diluted with 1% BSA inF12 were added to the wells, allowed to bind for 2 hours at 4° C., andwashed 3× with BSA/TBS at room temperature. Monoclonal antibodiesdiluted in BSA/TBS were incubated for 90 minutes at room temperature anddetected with a VECTASTAIN® ABC kit to mouse IgG. The HRP label wasvisualized with BIO-RAD® peroxidase substrate and read at 405 nm on aDynex MRX-TC microplate reader.

EXAMPLE 2 Development and Characterization of Anti-ADDL Antibodies

Three mice were inoculated with ADDLs (194±25 μg protein/injection)every three weeks for a total of six inoculations. Hybridomas made fromthe fusion of these mice spleens with SP2 cells were grown in 96-wellplates. Supernates from these wells were screened in dot blots withsynthetic ADDLs to identify positive clones, which were compared withdot blots of endogenous fibrils to identify differences. Hybridomas thatbound only synthetic ADDLs and not endogenous fibrils were sought. Tofurther refine what the products of the hybridomas bound to and underwhat conditions binding occurred, three western blots of each positiveclone were performed: SDS-PAGE of ADDLs, native gels of ADDLs, andSDS-PAGE with endogenous fibrils. Approximately 40 clones were selectedfor further examination. Each clone was tested for recognition ofsoluble Alzheimer's Disease brain extract, for identification of ADDLsbound to cultured hippocampal cells, and for the ability to block ADDLbinding under various conditions. Selected antibodies were collectedfrom culture medium and further purified using Protein G SEPHAROSE™.

Each time a set of hybridomas was screened via dot blot, approximately˜30% yielded positive supernates. Of these, only one or two hybridomasbound synthetic ADDLs and not endogenous fibrils. Approximately 2% ofthe original number of clones bound synthetic ADDLs and not monomer atlow ADDL concentrations, as determined by western blot analysis. Clone3B7, which bound synthetic ADDLs and not fibrils on western blots, waskept for further analysis.

One to two clones were identified that bound higher molecular weightmaterial (12-24 mer) better than trimer/tetramer oligomers. Two to threeclones were identified which could bind to native ADDLs under nativeconditions, but failed to bind ADDLs in the presence of SDS.

The results of this analysis indicated that ADDLs are good antigens inmice and monoclonal antibodies can be developed that bind to syntheticADDLs with much greater affinity than to monomers.

EXAMPLE 3 Immunohistochemical Analysis of Endogenous and Synthetic ADDLsBound to Cultured Hippocampal Cells

Cultured hippocampal cells were also analyzed to determine whethermonoclonal antibodies that distinguish between Alzheimer's Disease andcontrol brain extracts could identify ADDLs (either endogenous orsynthetic) bound to cultured cells. Hippocampal cultures were preparedaccording to established protocols and allowed to grow for 3-4 weeks.Synthetic ADDLs were prepared according to standard protocols (e.g.,U.S. Pat. No. 6,218,506). Endogenous ADDLs were extracted fromAlzheimer's Disease brain according to Gong, et al. ((2003) supra).ADDLs (100 nM in F12, or 2 mg total protein in F12) were incubated withthe cells for 1 hour and then washed and fixed according to standardmethods. Following washing, the cells were incubated with 20C2, 3B7,M94, 2A10, 4E2, 2D6, 4C2, 2B4, 5F10, or 5G12 monoclonal antibody andsubsequently with anti-mouse secondary conjugated to ALEXAFLUOR® 488.Images were taken on a NIKON® DIAPHOT™ epifluorescent microscope withCOOLSNAP™ HQ camera and analyzed using METAMORPH™ software (UniversalImaging, Downingtown, Pa.).

Both endogenous and synthetic ADDLs exhibited the standard hot spotpattern in cultured cells when visualized by 20C2. Thus, monoclonalantibody 20C2 identifies both synthetic and endogenous ADDLs bound tocultured hippocampal cells. As 3B7 did not bind to fibrils, highermolecular weight oligomers, and monomers, hot spot binding of ADDLs by3B7 was attributed to oligomeric ADDLs. The other antibodies appeared torecognize a variety of epitopes on ADDLs bound to cells, ranging fromhot spots on processes (M94, 2A10) to cell body specific attachment(4E2) and other states in between (2D6, 4C2, 2B4, 5F10, 5G12).

EXAMPLE 4 Inhibition of ADDL Binding to Neurons Using Murine Anti-ADDLAntibodies

To determine whether monoclonal antibodies that distinguish betweenAlzheimer's Disease and control brain extracts could also block bindingof ADDLs to cultured cells, cultured hippocampal cells were preincubatedwith 20C2 antibody and ADDL binding was determined byimmunocytochemistry. Hippocampal cultures were prepared according toestablished methods and allowed to grow for 3-4 weeks. Synthetic ADDLswere prepared according to standard protocols (e.g., see U.S. Pat. No.6,218,506 and the like). Endogenous ADDLs were extracted fromAlzheimer's Disease brain according to Gong, et al. ((2003) supra).ADDLs (100 nM in F12, or 2 mg total protein in F12) were preincubatedwith 20C2 antibody for 1 hour and subsequently added to cells for 1 hourat 37° C. Cells were washed, fixed, and incubated with anti-mousesecondary conjugated to ALEXAFLUOR® 488.

Both endogenous and synthetic ADDL binding to cultured cells was blockedby preincubation with 20C2. Vehicle and no-secondary antibody controlimages were black.

EXAMPLE 5 Detection of ADDL Binding to Neurons Using Biotinylated ADDLs

The binding of ADDLs or bADDLs (biotinylated ADDLS) to neurons wasdetected using standard immunofluorescence procedures. Primaryhippocampal neurons (cultured for fourteen days) or B103 cells (platedfor twenty-four hours) were incubated with 5-25 μm ADDLs or bADDLs forone hour at 37° C. and the cells were subsequently washed three to fourtimes with warm culture medium to remove unbound ADDLs or bADDLs. Thecells were then fixed for ten minutes at room temperature with 4%paraformaldehyde prepared from 16% paraformaldehyde (Electron MicroscopySciences, Fort Washington, Pa.) diluted in PBS. Subsequently, thesolution was removed and fresh fixative added for an additional tenminutes at room temperature. The cells were permeabilized (4%paraformaldehyde solution with 0.1% TRITON™-X 100; SIGMA, St. Louis,Mo.) for ten minutes, washed six times with PBS and incubated for onehour at 37° C. with blocking buffer (PBS with 10% BSA; Sigma, St. Louis,Mo.). At this point, the protocols for the detection of bound ADDLs andbADDLs diverge. To detect ADDL binding, the cells were incubatedovernight at 37° C. with 4G8 (diluted 1:1,000 in PBS containing 1% BSA;Signet Labs, Dedham, Mass.), 6E10 (1:1,000; Signet Labs, Dedham, Mass.),or one of the anti-ADDL monoclonal antibodies disclosed herein (diluted1:1,000). In addition, a polyclonal antiserum raised against tau(1:1,000; Sigma, St. Louis, Mo.) was used to visualize the cellprocesses. The next day, the cells were washed three times with PBS,incubated for one hour at room temperature with an ALEXA® 594-labeledanti-mouse secondary (diluted 1:500 in PBS with 1% BSA; MolecularProbes, Eugene, Oreg.) and an ALEXA® 488-labeled anti-rabbit secondary(diluted 1:1,000; Molecular Probes, Eugene, Oreg.), washed three timesin PBS and the binding observed using a microscope with fluorescencecapabilities. For the detection of bADDL binding, the cells wereincubated overnight with the tau antibody. Subsequently, the cells werewashed three times with PBS, incubated for one hour at room temperaturewith an ALEXA® 488-labeled anti-rabbit secondary (as above) and anALEXA® 594-labeled streptavidin, 1:500 dilution (Molecular Probes,Eugene, Oreg.), washed 5-6 times in PBS and the binding visualized witha fluorescence microscope. If the staining of the cell nuclei wasdesired, the nuclei were labeled with DAPI (1:1000) according tostandard protocols.

For immunocytochemical analysis of ADDLs using an ADDL-specificmonoclonal antibody, cells were washed, fixed, permeabilized and blockedafter incubation with ADDLs. To detect the bound bADDLs with monoclonalantibodies, the cells were incubated overnight with 4G8, 6E10 or one ofthe instant anti-ADDL monoclonal antibodies and immunoreactivity wassubsequently detected with an ALEXA® 488-labeled anti-mouse secondaryantibody. The bound bADDLs were visualized with an ALEXA® 594-labeledstreptavidin and the nuclei stained with DAPI. After staining, thecolocalization of bADDL binding and ADDL immunoreactivity was detectedwith a fluorescence microscope.

Specific immunoreactivity with primary hippocampal cells incubated withADDLs was seen with each of the monoclonal antibodies evaluated (i.e.,20C2, 2H4, 2B4, and 2A10). The bound ADDLs appeared as punctate stainingalong the neuronal processes and cell soma. This pattern was only seenon a subset of neurons, a pattern that is consistent with previousreports describing ADDL binding to primary neurons using both commercialand non-commercial antibodies. The pattern of staining and the resultsof a number of control studies demonstrated the specificity of theseantibodies.

The use of bADDLs offered a simplified method to detect bound ADDLs andevaluate the blockade of ADDL binding with the monoclonal antibodies.When bADDLs were added to primary hippocampal cells and the bindingevaluated with a fluorescent-labeled streptavidin, specific binding wasseen along the neuronal processes of a subset of cells in culture. Ifthe cells were then fixed, processed for immunocytochemistry and ananti-ADDL antibody used to visualize binding, a similar pattern ofstaining was observed. Furthermore, the superimposition of thesestaining patterns revealed a perfect overlap of the antibody stainingand bound bADDLs, thus demonstrating that bADDLs and ADDLs arefunctionally equivalent and the use of bADDLs in binding assays.

EXAMPLE 6 Detecting and Measuring Murine anti-ADDL Monoclonal AntibodyDifferential Displacement of bADDL Binding to Neurons

The ability of antibodies to block the binding of ADDLs or bADDLs toneuronal cultures (primary neurons or B103 cells) was characterizedusing the immunocytochemical methods described herein with a fewmodifications. Monoclonal antibodies were mixed with 1-10 μm bADDLs at amolar ratio of 1:1, 1:5 or 1:10 (antibody:bADDLs) and incubated in asiliconized microcentrifuge tube for one hour at 37° C. on a slowrotator (Miltenyi Biotec, Auburn, Calif.). Subsequently, theantibody/bADDL mixture was added to cells and allowed to furtherincubate for one hour at 37° C. After incubation, the cells were washed,fixed, permeabilized, blocked and incubated overnight with a polyclonalantiserum raised against tau to visualize the cell processes. The nextday, the cells were washed, incubated with an ALEXA® 488-labeledanti-rabbit secondary antibody and an ALEXA® 594-labeled streptavidinand the cells were stained with DAPI to allow detection of nuclei. Oncestained, the degree of binding was assessed visually with a fluorescencemicroscope.

To quantitatively assess the degree of bADDL binding and the ability ofanti-ADDL antibodies to abate this interaction, a cell-based alkalinephosphatase assay was developed. Monoclonal antibodies or PBS were mixedat a 1:1 (B103 cells) or 1:5 (primary neurons) molar ratio with 2.5-10μm (final concentration) of bADDLs and incubated for one hour at 37° C.on a slow rotator. After preincubation, the antibody/bADDL preparationswere added to the B103 or primary neuron cultures and incubated for anadditional one hour at 37° C. At the end of the incubation period, thebADDLs/antibody mixture was removed and the plates washed six times withmedia. The cells were fixed in 4% paraformaldehyde for ten minutes atroom temperature, the solution removed, fresh fixative added and thecells fixed for an additional ten minutes. The cells were permeabilizedwith 4% paraformaldehyde containing 0.1% TRITON™ X-100 (2 times, eachfor ten minutes at room temperature), washed six times in PBS andtreated with 10% BSA in PBS for one hour at 37° C. Alkalinephosphatase-conjugated streptavidin (1:1,500 in 1% BSA; MolecularProbes, Eugene, Oreg.) was added to the cells for one hour at roomtemperature. The cells were rinsed six times with PBS, the alkalinephosphatase substrate (CDP-STAR® with SAPPHIRE-II™; Applied Biosystems,Foster City, Calif.) added to the cells and incubated for thirty minutesprior to determining the luminescence on a LJL Luminometer (Analyst AD;LJL BioSystems, Sunnyvale, Calif.).

When the binding of bADDLs to the neurons was evaluated, anantibody-dependant pattern of staining was observed. Some of theantibodies investigated markedly reduced the binding of bADDLs, whileothers were less effective. Unexpectedly, a third group of antibodiesappeared to enhance the binding of bADDLs to neurons. While the resultsof these studies were qualitative and not quantitative in nature, theyindicated that the antibodies differentially blocked bADDL binding toneurons. Quantitative assessment demonstrated a similar trend (FIG. 1).That is, some antibodies abated the binding of bADDLs to neurons, somewere weak or had little effect and a few enhanced the binding (i.e.,5F10 and 4C2). Moreover, a mouse Fab was unable to block the binding ofbADDLs, further demonstrating the specificity of the monoclonalantibodies in this assay.

Analysis of bADDL binding and blockade with monoclonal antibodies in theneuroblastoma cell line B103 demonstrated specific bADDL binding to B103cells, but not to an ovarian cell line (CHO). Moreover, the binding wasdramatically attenuated when bADDLs were pre-incubated with an anti-ADDLmonoclonal antibody prior to the addition to B103 cells. Quantitativeassessment of the blockade of bADDL binding to B103 cells withmonoclonal antibodies indicated that the monoclonal antibodies were notequal in their ability to block bADDL binding to cells (FIG. 2). As seenwith the primary hippocampal cells, some antibodies were quite good atblocking binding, while others were less effective. Furthermore, theantibody 4C2 also enhanced the ability of bADDLs to bind to B103 cellsin culture.

To show that bADDLs also bind to regions of the hippocampus that areinvolved in learning and memory, a series of binding studies wereconducted using rat hippocampal slice cultures. Binding studies showedthat neurons in the CA1-3 and dentate gyrus regions of the hippocampuswere capable of binding bADDLs, while neurons in other regions did not.When the bADDLs were pre-incubated with an anti-ADDL monoclonalantibody, the degree of bADDL binding was attenuated in a dose-dependantmanner. These results showed that monoclonal antibodies can also abatethe binding of bADDLs to a subset of hippocampal neurons, neurons that acritical for learning and memory.

EXAMPLE 7 Binding of Anti-ADDL Antibodies to Endogenous ADDLs fromAlzheimer's and Control Brain

To further characterize the monoclonal antibodies disclosed herein, itwas determined whether the monoclonal antibodies could identify ADDLsfrom soluble extracts of human Alzheimer's Disease brain (endogenousADDLs) and distinguish that from extracts of control brain. SyntheticADDLs and human brain extracts prepared in F12 were diluted in F12 andspotted (1 pmole ADDLs; 0.5 μg brain extract) in duplicate onto dryHYBOND™ ECL™ nitrocellulose. Brain tissue, with corresponding CERADgrades (Consortium to Establish a Registry for Alzheimer's Disease) andBraak stages, was obtained from NU Brain Bank Core. The blot was allowedto dry 20 minutes and then incubated in 3% H₂O₂ in TBS (20 mM Tris-HCl,pH 7.5, 0.8% NaCl) for 20 minutes at room temperature. The blot was cutinto strips and blocked with 5% milk in TBS-T (0.1% TWEEN™-20 in TBS)for 1 hour at room temperature. Rabbit polyclonal antibody M71/2(1:2500, 0.4 μg; Bethyl Laboratories, Inc., Montgomery, Tex.);monoclonal antibody 6E10 (1:500, 3 μg; Signet Labs, Dedham, Mass.); andmonoclonal antibodies 20C2 (1.52 mg/mL, 5 μg), 11B5 (2.54 mg/ml, 5 μg),2B4 (1.71 mg/mL, 5 μg), and 2A10 (1.93 mg/mL, 7.5 μg) as disclosedherein (FIG. 3) were diluted in 1.5 mL of milk/TBS-T and incubated for 1hour at room temperature. The blots were washed 3×10 minutes with TBS-T.The blots were incubated with horseradish peroxidase (HRP)-linkedsecondary antibody (1:40,000 in milk/TBS-T; Amersham Life Science, Inc.,Arlington Heights, Ill.) for 1 hour at room temperature. The blots werewashed 3×10 minutes with TBS-T, rinsed 3 times with dH₂O, developed withSUPERSIGNAL™ substrate (1:1 dilution with ddH₂O; Pierce, Rockland, Ill.)and exposed to HYPERFILM™ ECLT™ (Amersham Life Science, Inc., ArlingtonHeights, Ill.).

All antibodies tested identified synthetic ADDLs with robust binding,except 2A10, which had weaker binding, even though it was tested athigher protein concentration. Polyclonal antibody M71/2 and monoclonalantibodies 20C2 and 11B5 bound strongly to both Alzheimer's Diseasesamples, but showed only very faint binding, similar to background incontrol brain. In contrast, monoclonal antibodies 6E10, 2B4, and 2A10showed weak binding to Alzheimer's Disease brain.

The results of this analysis indicated that two of the monoclonalantibodies tested could distinguish between Alzheimer's Disease andcontrol brain, wherein binding to endogenous oligomers was with a highdegree of specificity. In addition, these data indicate that detectioncan be accomplished in early stages of Alzheimer's Disease.

EXAMPLE 8 Immunohistochemical Analysis of Alzheimer's Disease andControl Brain Slices

Immunohistochemical analysis using the monoclonal antibodies disclosedherein was carried out to determine whether ADDLs can be visualized inbrain slices using monoclonal antibodies that distinguish betweenAlzheimer's Disease and control brain extracts, and to demonstrate thenature of ADDL labeling (e.g., diffuse, perineuronal, plaque-like, etc.)and its distribution in human tissue. Sections (40 μm) of fixedAlzheimer's Disease and control brain were prepared in accordance withstandard methods. The slices were labeled with several monoclonal andone polyclonal antibody and subsequently counterstained with hematoxylinto identify cell nuclei. Images were obtained using a NIKON® ECLIPSE®E600 light microscope with a SPOT™ INSIGHT™ digital video camera (v.3.2).

Immunohistochemical analysis indicated that ADDL staining was manifestin Alzheimer's Disease brain in the hippocampus, entorhinal cortex, andmiddle frontal gyrus. In a severe Alzheimer's Disease case, there wasabundant light ADDLs staining in what appeared predominantly as aplaque-type distribution. Some light ADDL staining was observed asperi-neuronal in one Alzheimer's Disease case. In contrast, there is nostaining using either antibody in any regions of control samples, noteven a rare neuron surrounded by dot-like immunostaining.

These data indicate that polyclonal and monoclonal antibodies can beused to identify ADDLs in fixed human tissue, wherein labeling isvaried, consisting of plaque-like regions, vascular regions, andperi-neuronal labeling of individual cells and some clusters. Further,labeling of ADDLs in Alzheimer's Disease, but not control, brain wasobserved in at least three brain regions: hippocampus, entorhinalcortex, and middle frontal gyrus.

EXAMPLE 9 Immunostaining of Aβ1-40 Monomer-Like Control

Aβ1-40 oligomerizes slowly in DMSO/F12 compared to ADDLs. Thus, it wasdetermined whether Aβ1-40 could serve as a monomer-like control. ADDLswere subjected to size exclusion chromatography (SEC) on a SUPERDEX® 75column (ADDL063), which resolved into two peaks. Aβ1-40 was prepared inDMSO/F12 (45.5 mM), frozen and thawed. Samples were diluted with F12 andmixed ˜2:1 with Tricine sample buffer (BIO-RAD®, Waltham, Mass.).SDS-PAGE was carried out on 10-20% Tris-Tricine gels (BIO-RAD®, Waltham,Mass.) with Tris/Tricine/SDS buffer (BIO-RAD®, Waltham, Mass.) at 120 Vat room temperature for 80 minutes. The gel was silver stained (60pmoles Aβ1-40 or ADDLs; 40 pmoles Peaks 1 or 2) with SILVERXPRESS™(INVITROGEN™, Carlsbad, Calif.). Alternatively, the gels (20 pmolesAβ1-40 or ADDLs; 30 pmoles Peaks 1 or 2) were electroblotted ontoHYBOND™ ECL™ nitrocellulose using 25 mM Tris-192 mM glycine, 20% v/vmethanol, pH 8.3, 0.02% SDS at 100 V for 1 hour at 8° C. The blots wereblocked with 5% milk in TBS-T (0.1% TWEEN™-20 in 20 mM Tris-HCl, pH 7.5,0.8% NaCl) overnight at 8° C. Monoclonal antibody 6E10 (1:2000; SignetLabs, Dedham, Mass.), monoclonal antibody 20C2 (1.52 mg/mL, 1:2000; FIG.3), or polyclonal antibody M71/2 (1:4000, Bethyl Laboratories, Inc.,Montgomery, Tex.) was diluted in milk/TBS-T and incubated with the blotsfor 90 minutes at room temperature. The blots were washed 3×10 minuteswith TBS-T and subsequently incubated with HRP-conjugated secondaryantibody (1:40,000 in TBS-T; Amersham Life Science, Inc., ArlingtonHeights, Ill.) for 1 hour at room temperature. After three washes withTBS-T, 10 minutes per wash, the blots were rinsed 3× with dH₂O,developed with SUPERSIGNAL® West Femto Maximum Sensitivity substrate(1:1 dilution with ddH₂O; Pierce, Rockland, Ill.) and exposed toHYPERFILM™ ECL™ (Amersham Life Science, Inc., Arlington Heights, Ill.).

Silver stain analysis showed Aβ1-40 as a heavy monomer band. Incontrast, ADDLs and Peak 1 showed monomer, trimer and tetramer, althoughthere was less tetramer. Silver stain analysis of Peak 2 showed heavymonomer with a lighter trimer and very light tetramer band.

Immunostaining of Aβ1-40 with 6E10 showed only a light monomer band.Immunostaining of ADDLs and Peak 1 with 6E10 showed monomer, trimer,tetramer and 12-24mer. Peak 2 showed heavy monomer staining with 6E10and some light trimer and tetramer with no 12-24mer. There was nomonomer staining of Aβ1-40 with 20C2 or M71/2. While both 20C2 and M71/2showed minimal or no monomer staining of ADDLs and Peak 1, these sampleshad trimer, tetramer, and 12-24mer staining with 20C2 and M71/2. Peak 2immunostaining with 20C2 and M71/2 showed light monomer, trimer andtetramer with no 12-24mer observed. Aβ1-40 immunostained lighter with6E10 than did the ADDL monomer, despite heavier silver staining.

These results indicated that, in contrast to the 6E10 antibody whichshows good recognition of monomer, gels transferred with 0.02% SDS inthe transfer buffer showed minimal monomer detection with theoligomer-specific antibodies. Immunostaining of SEC fractions showedPeak 2 composed mostly of monomer with small amounts of trimer andtetramer and no 12-24mer, while Peak 1 has monomer, trimer, tetramer andthe 12-24mers.

To further characterize the monoclonal antibodies with respect tobinding to Peak 1 and Peak 2, a sandwich ELISA was developed usingpolyclonal antibody M90 to ADDLs as the capture antibody. SEC peak 1 andpeak 2 fractions referred to herein are the two major peaks of ADDLsthat were fractionated on a SEPHADEX™ 75 column to distinguish betweenpotentially bioactive and inactive oligomers. Non-denaturing gelelectrophoresis confirmed the separation into large (>50 kDa) and small(<30 kDa) aggregates that were stable at 37° C. These peaks were usedseparately as the detection substance for clone supernates. Binding wasvisualized with a VECTASTAIN® kit. Differences between recognition ofthe two peaks was observed for all antibodies. For example, compare theratio of peak 1 to peak 2 for antibodies 2B4 and 20C2 (FIG. 3). Only oneantibody reflects the control antibody (6E10) preference for peak 2.

EXAMPLE 10 Detection of ADDL Formation from Aβ1-42

Polyclonal antibodies have been used in dot-blots to show time-dependentADDL formation from Aβ1-42. Thus, it was demonstrated that monoclonal20C2 antibody, which preferentially binds to oligomers, could also showincreased signal with time as ADDLs form from Aβ1-42. Aβ1-42, ˜750pmoles HFIP film, was dissolved in 1.5 mL DMSO (0.5 mM) and 2 μLaliquots diluted to a final volume of 100 μL with F12 (10 nM) andincubated on ice. Two μL (20 fmol) of reaction mixture was spotted ondry HYBOND™ ECL™ nitrocellulose (Amersham Life Science, Inc., ArlingtonHeights, Ill.) at specified time points. The nitrocellulose was blockedwith 5% non-fat dry milk in TBS-T (20 mM Tris-HCl, pH 7.5, 0.8% NaCl,0.1% TWEEN™-20) for 1 hour at room temperature. Polyclonal antibodyM90/1 (Bethyl Laboratories, Inc., Montgomery, Tex.) or monoclonalantibody 20C2 (1.52 mg/mL) was diluted 1:2000 in milk/TBS-T andincubated with the blot for 90 minutes at room temperature followed bywashing 3×10 minutes with TBS-T. HRP-conjugated secondary antibodies(Amersham Life Science, Inc., Arlington Heights, Ill.) were diluted1:40,000 in milk/TBS-T and the blot incubated for 60 minutes at roomtemperature followed by washing as above. After a brief rinse with dH₂O,the blot was incubated for 60 seconds with SUPERSIGNAL® West FemtoMaximum Sensitivity substrate (diluted 1:1 with ddH₂O; Pierce, Rockland,Ill.) and exposed to HYPERFILM™ ECL™ (Amersham Life Science, Inc.,Arlington Heights, Ill.). Dot blots were scanned and intensity of spotswas determined with ADOBE® PHOTOSHOP®.

Both antibodies detected time-dependent ADDL formation from Aβ1-42,wherein the results for 20C2 showed better signal and consistency.Neither antibody could detect Aβ1-40 at a concentration equivalent toADDLs. These data further demonstrate the oligomer-specificity of thisantibody, since monomers are present all the time and oligomers formwith time. In addition, both M90/1 and 20C2 showed minimal recognitionof Aβ1-40 monomers even at a 100-fold higher concentration than ADDLs.

EXAMPLE 11 Competition Dot Blot Assays

To determine whether the monoclonal antibodies disclosed herein couldbind monomers, a competition dot blot assay was performed with syntheticADDLs, 20C2, and Aβ1-40. ADDLs were applied to dry nitrocellulose at 10μmol/0.5 μL. While the nitrocellulose was being blocked in 5% NDM/TBS-Tfor one hour, ADDLs and fresh Aβ1-40 at various concentrations wereincubated with 200 μL each of 20C2 (1.5 μg/mL final concentration) in 5%NDM/TBS-T for 1 hour. These solutions were then applied to thenitrocellulose using the SURF-BLOT apparatus and incubated at roomtemperature for 1.5 hours with rocking. The blot was subsequentlyvisualized with anti-mouse IgG-HRP and chemiluminescence. Quantitationwas performed using the KODAK® IMAGESTATION® 440 and EXCEL®.

Results of this analysis indicated that synthetic ADDLs in solutioncould effectively and specifically block 20C2 binding to ADDLsimmobilized on nitrocellulose with a half maximal inhibition observed at<50 nM for ADDLs. In contrast, Aβ1-40 in solution did not block bindingof 20C2 to immobilized ADDLs.

To determine which portions constitute the binding epitope of the Aβ1-42molecule, a competition dot blot assay was performed with ADDLs, 20C2,and peptides. ADDLs were spotted on nitrocellulose at fourconcentrations (1, 0.5, 0.25, and 0.125 pmole) each in 0.5 μL. While thenitrocellulose was being blocked in 5% NDM/TBS-T for two hours, thepeptides at 50, 100 and 200 μmol were added to 200 μL of 20C2 (1.52μg/mL final concentration=1.9 μmol, in 5% NDM/TBS-T) and rocked at roomtemperature. The solutions were subsequently incubated with thenitrocellulose using the SURF-BLOT apparatus for 1.5 hours at roomtemperature. Binding was visualized with anti-mouse IgG-HRP usingchemiluminescence.

The results of this analysis indicated that binding to ADDLs was blockedby the ADDLs themselves and by Aβ1-28, but no other combination ofpeptides. Thus, the binding epitope required some conformation thatAβ1-28 could attain, but that was not available on Aβ1-12 and Aβ12-28 ortheir combination. Alternatively, Aβ1-28 forms a dimer that blocksbinding of ADDLs by steric hindrance.

To determine whether Aβ1-28 aggregates (similar to Aβ1-42) or folds suchthat it blocks the binding epitope for 20C2, SDS-PAGE gels were silverstained and western blot analysis was performed. ADDLs and Aβ1-28 (60pmol in each of two lanes used for silver stain and 20 pmol otherwise)were separated using a 10-20% Tris-Tricine SDS-PAGE. The 60 pmol laneswere excised and stained with SILVERXPRESS™ (INVITROGEN™, Carlsbad,Calif.); alternatively, the gels (20 pmoles ADDLs and Aβ1-28) wereelectroblotted onto HYBOND™ ECL™ nitrocellulose using 25 mM Tris-192 mMglycine, 20% v/v methanol, pH 8.3, 0.02% SDS at 100V for 1 hour at 8° C.The blots were blocked with 5% milk in TBS-T (0.1% TWEEN™-20 in 20 mMTris-HCl, pH 7.5, 0.8% NaCl). Samples were incubated with 20C2 (1:1000,1.52 mg/mL) or 20C2+Aβ1-28 (2 nmol, preincubated for 2 hour) for 1.5hour at room temperature in the above blocking buffer. Binding wasvisualized with anti-mouse IgG-HRP (1:40,000 in TBS-T) andchemiluminescence.

Silver staining showed monomer, trimer and tetramer in the ADDL lane,whereas the Aβ1-28 lane had one species, which ran at about a dimer.ADDLs, but not Aβ1-28, were visualized by 20C2 and binding to all ADDLspecies by 20C2 was blocked by Aβ1-28. Moreover, while the 20C2 bindingepitope is blocked by Aβ1-28, 20C2 does not recognize the Aβ1-28 peptidein a western blot.

EXAMPLE 12 Isotype Analysis of Anti-ADDL Antibodies

To further characterize the monoclonal antibodies disclosed herein,isotype analysis was performed using the SIGMA IMMUNOTYPE™ Kit with theMouse Monoclonal Antibody Isotyping Reagents, following themanufacturer's directions (Sigma-Aldrich Co., St. Louis, Mo.). Resultsof this analysis are presented in FIG. 3.

EXAMPLE 13 Core Linear Epitope Mapping of Anti-ADDL Antibodies

Specific interaction of the anti-ADDL monoclonal antibodies with amyloidbeta peptide was detected in standard ELISA assays. Briefly, syntheticpeptides, or ADDL or fibril in some cases, were used as antigen to coaton NUNC™ MAXISORB™ plate at concentration of 4 μg/mL (about 800 to 1200nM). Unless specified, the peptides were coated in 5 mM sodiumbicarbonate buffer, pH 9.6, overnight at 4° C. After blocking the plateswith PBS containing 0.05% TWEEN™ 20 and 3% (w/v) nonfat dry milk for onehour, the monoclonal antibody was titrated in blocking buffer at adetermined concentration and the plates were incubated for one hour atambient temperature with gentle rocking. After washing, HRP-conjugatedgoat anti-mouse IgG (H+L), diluted in blocking buffer, was added to theplates. The calorimetric substrate, TMB, was added to the plates afterextensive washes to remove unbound HRP-conjugate. The absorbance wasmeasured at wavelength of 450 nm on a plate reader.

To map the core linear epitope for the anti-ADDL monoclonal antibodies,a set of overlapping, ten amino acid peptides was synthesized to coverAβ1-42 (Table 1). Three peptides of fourteen amino acids, with reversedamino acid sequence of Aβ1-42 were also synthesized as nonspecificcontrol peptides.

TABLE 1 SEQ Mol. ID N- C- Pepticle Sequence Wt. NO: 1 42DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 180 1 7 DAEFRHD 929.8 181 1 8DAEFRHDS 975.4 178 1 9 DAEFRHDSG 10352.5 182 1 10 DAEFRHDSGY 1195.4 1832 11  AEFRHDSGYE 1209.3 184 3 12   EFRHDSGYEV 1237.4 185 4 13   FRHDSGYEVH 1245.2 186 5 14     RHDSGYEVHH 1235.7 187 6 15     HDSGYEVHHQ 1207.4 188 7 16       DSGYEVHHQK 1198.5 189 8 17       SGYEVHHQKL 1196.8 190 9 18         GYEVHHQKLV 1208.3 191 10 19         YEVHHQKLVF 1298.6 192 11 20           EVHHQKLVFF 1282.9 193 1221            VHHQKLVFFA 1224.4 194 13 22             HHQKLVFFAE 1254.5195 14 23              HQKLVFFAED 1232.5 196 15 24              QKLVFFAEDV 1177.3 197 16 25                KLVFFAEDVG1123.8 198 17 26                 LVFFAEDVGS 1082.3 199 18 27                 VFFAEDVGSN 1083.0 200 19 28                  FFAEDVGSNK 1112.2 201 20 29                   FAEDVGSNKG 1022.6 202 21 30                    AEDVGSNKGA 946.5 203 22 31                     EDVGSNKGAI 988.1 204 23 32                      DVGSNKGAII 972.2 205 24 33                       VGSNKGAIIG 914.4 206 25 34                        GSNKGAIIGL 928.5 207 26 35                         SNKGAIIGLM 1002.2 208 27 36                          NKGAIIGLMV 1014.7 209 28 37                           KGAIIGLMVG 957.4 210 29 38                            GAIIGLMVGG 886.3 211 30 39                             AIIGLMVGGV 928.3 212 31 40                              IIGLMVGGVV 956.5 213 32 41                               IGLMVGGVVI 956.4 214 33 42                                GLMVGGVVIA 914.2 215 14 1 HHVEYGSDHRFEAD1923.8 216 28 15               KNSGVDEAFFVLKQ 1806.9 217 42 29                            AIVVGGVMLGIIAGKK 1751.5 218

All peptides were dissolved in DMSO at about 400 to 500 μM (1 mg/mL) andstored in multiple aliquots at −20° C. The peptides were used in anELISA assay for determination of the core epitope of the anti-ADDLmonoclonal antibodies. Each monoclonal antibody was tested at fourconcentrations (3, 1, 0.3 and 0.1 μg/mL) against either an N-terminalpeptide set (from residues 1 to 25) or a C-terminal peptide set (fromresidues 17 to 42), with control peptides. The core linear epitopes forthe panel of monoclonal antibodies are listed in Table 2. Severalcommercial monoclonal antibodies (6E10, BAM-10, 4G8 and WO-2) wereincluded in the experiment to validate the assay format, and the resultsconfirmed their core linear epitopes as reported in publishedliterature.

TABLE 2 SEQ Core ID Antibody Epitope* Epitope Sequence within Aβ1-42 NO:DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 180 6E10  5-11     RHDSGYE219 BAM-10 3-8   EFRHDS 220 4G8 xx-21           EVHHQKLVFFA 221 WO-2 3-8  EFRHDS 220 26D6 3-8   EFRHDS 220 2A10^(a) 3-8   EFRHDS 220 2B4^(b) 3-8  EFRHDS 220 4C2^(a) 3-8   EFRHDS 220 4E2^(a) 3-8   EFRHDS 220 2H4^(c)1-8 DAEFRHDS 178 20C2^(a) 3-8   EFRHDS 220 2D6^(a) 3-8   EFRHDS 2205F10^(c) 3-8   EFRHDS 220 1F4^(a) nd 1F6^(a) nd 2E12^(a)  3-10  EFRHDSGY 222 3B3^(a) nd *Position within Aβ1-42. ^(a)IgG1, ^(b)IgG2b,^(c)IgG2a. nd, not determined.

Nine out of twelve ADDL-specific monoclonal antibodies evaluated weremapped to the N-terminal region of Aβ1-42, and seven of these mapped toamino acid residues 3 to 8. Two monoclonal antibodies, 2H4 and 2E12,prefer slightly bigger epitopes. Three monoclonal antibodies, 1F4, 1F6and 3B3, failed to bind the overlapping peptide set, even at highconcentration of 3 μg/mL, but their epitopes were estimated to belocated at the N-terminus of Aβ1-42, as they could bind to Aβ1-20peptide, which was used as a positive control in the experiments.

EXAMPLE 14 Affinity and Specificity of Mouse Anti-ADDL Antibodies

A solution-based binding assay was developed to determine thespecificity and affinity of anti-ADDL antibodies to different amyloidbeta peptide preparations (ADDL, fibril, Aβ1-40, Aβ1-20). A quantitativeELISA was established that was capable of capturing the linear range ofdose-response of monoclonal antibodies against ADDL coated on NUNCT™plates. Based on this information, a fixed concentration of monoclonalantibody was selected that could give consistent OD signals in ELISAjust above assay noise (OD 450 nm reading around 0.2 to 0.5). IgG atthis fixed concentration was then incubated with different amyloid betapeptide substrates (ADDL, fibril, Aβ1-40, Aβ1-20) in 20 point titrationsin solution at room temperature overnight to reach equilibrium. Thequantity of free IgG within the mixture was determined the next day in aquantitative ELISA with a one hour incubation on regular ELISA plates.The fraction of bound IgG was calculated and the correlations of boundIgG to titration of free ligand (substrates) were used to derive K_(D),using the GraFit program (Erithacus Software, Surrey, UK). Thus, thesubstrate preference for each antibody to different amyloid beta peptidepreparations was presented as the intrinsic affinity values (K_(D)).

There were several advantages of using this assay format. First, theinteraction of the antibody and substrate was in solution phase, thus,there was no constraint from any solid surface such as in regular ELISAassay or BIACORE™ experiment, where potential influence of solid surfacefrom ELISA plates or sensor chip on monoclonal antibody and substrateinteraction has to be taken into consideration for interpretation ofdata. Second, the interactions were allowed to reach equilibrium.Therefore, the interaction of IgG and substrate occurred at limitingconcentrations of both components with no concerns for precipitation ofIgG or additional amyloid beta peptide oligomerization due to highexperimental concentration. Third, the assay readout was independent ofantigen in the solution; thus, any heterology of amyloid beta indifferent peptide preparations (e.g., ADDL or fibril) would notinterfere with data interpretation and mathematical modeling. The assaysensitivity was limited to ELISA assay detection limits which allowedthis assay to evaluate monoclonal antibodies with K_(D) values in thenanomolar range. Alternative substrates such fluorescent reagents arecontemplated to improve the sensitivity range. It is believed that theimmune complex was minimally disrupted during the one hour incubation tocapture the free IgG in quantitative ELISA.

The quantities of free IgG were determined by a standard curve andplotted against titrations of different substrate. The quantities ofbound IgG with different substrates were plotted and the information wasused in GraFit for curve fitting with appropriate mathematic models. Thesummary of K_(D), expressed in nM ranges, for the panel of monoclonalantibodies disclosed herein is presented in Table 3.

TABLE 3 ADDL Fibril Aβ1-40 Aβ1-20 Antibody* K_(D) SE K_(D) SE K_(D) SEK_(D) SE 20C2 0.92 0.09 3.62 0.47 30.48 5.05 71.35 24.41 2A10 2.29 0.256.72 0.99 14.69 2.64 22.40 2.43 2B4 2.09 0.24 10.50 1.26 27.57 4.88 1.630.26 2D6 5.05 0.52 14.41 2.40 25.66 5.84 30.17 7.07 5F10 11.90 1.6328.95 5.78 23.54 6.21 6.10 4.39 4E2 4.26 0.42 9.40 1.60 20.24 2.07 28.403.23 4C2 8.08 1.03 19.17 3.69 21.89 4.14 28.40 3.23 1F4 9.24 0.84 12.521.66 IC IC IC IC 1F6 N/T N/T N/T N/T N/T N/T N/T N/T 3B3 10.02 0.74 7.210.59 104.68 21.86 IC IC 2E12 IC IC IC IC IC IC IC IC WO-2 0.57 0.0421.15 0.12 6.15 0.62 19.26 3.53 *All antibodies were IgG. Values listedin italic are high SE and poor fitting. IC: inconclusive data N/T: nottested.

EXAMPLE 15 Detecting and Measuring Tau Phosphorylation

Hyperphosphorylated Tau (pTau) is a hallmark of Alzheimer's Disease,although little is known about the events that cause thishyperphosphorylation. Without wishing to be bound by any theory, it isbelieved that ADDLs may play a role in this phosphorylation event. Toinvestigate this, neuronal cultures (primary neurons and B103 cells)were grown as described above, 1 μm bADDLs or vehicle was added to themedia and the cultures were maintained for an additional one, six ortwenty four hours. At the end of each incubation, the cells were washed,fixed, permeabilized, blocked and incubated overnight with a monoclonalantiserum raised against pTau (AT8, 1:500; Pierce, Rockland, Ill.). Thenext day, the cells were washed, incubated with an ALEXA® 488-labeledanti-mouse secondary antibody and an ALEXA® 594-labeled streptavidin andthe cells were stained with DAPI to allow detection of nuclei. The cellswere then assessed using a fluorescence microscope, with the degree ofpTau staining and correlation with bADDL binding being noted at eachtime-point.

The results of this analysis indicated that bADDL binding to B103 cellsincreased the level of pTau in the cellular processes, when comparedwith vehicle-treated cells. A similar change was also noted in primaryhippocampal cells. When cells were exposed to bADDLs for six hours, anincrease in pTau staining was observed in a subpopulation of cells,cells that also bound bADDL. A time-course study with B103 cells furtherinvestigated the modulation of pTau by bADDLs. The addition of bADDLsresulted in a marginal increase in pTau at one hour. However, pTaustaining was dramatically increased six hours after the addition ofbADDLs and remained elevated up to 24 hours later. Thus, these dataindicate that ADDL binding to neurons can initiate a cascade ofintracellular events that results in the hyperphosphorylation of tau,the accumulation of neurofibrillary tangles and eventual cell death. Tothis end, one skilled in the art can appreciate that blocking thebinding of ADDLs to neurons, would in turn prevent such downstreamevents and be beneficial for the treatment of amyloidogenic diseasesand/or tauopathies. Moreover, a better understanding of the signalingevents that are triggered by ADDL binding and result in pTau productionmay also elucidate additional pathways that are suitable targets for thedevelopment of novel therapeutics.

EXAMPLE 16 Aβ Peptide/ADDL-Antibody Interaction and Assembly Inhibition

Changes in ADDL assembly kinetics and oligomeric size, in the presenceof selected monoclonal antibodies disclosed herein were observed byfluorescence resonance energy transfer (FRET) and fluorescencepolarization (FP) using a 1:4 mixture of fluorescein-labeled Aβ1-42monomers to native peptide monomers. The auto-quenching of flouresceinemission upon monomer incorporation into ADDLs results in a three- tofive-fold reduction of fluorescence intensity over the short hourtimescale due to FRET. In addition, the increase in size when monomersassemble into oligomeric ADDL species results in a two-fold FP increase.The FRET and FP kinetic progress curves of ADDL assembly, in thepresence of various novel and commercial anti-ADDL and anti-Aβ peptideantibodies, showed differences in the ability of the antibodies toinhibit ADDL assembly and/or bind peptide oligomers (FIG. 4).

Assays were performed in 384-well CORNING® Non-Binding Surface black,opaque microtiter plates. The assay buffer was composed of 50 mMMOPS-Tris (pH 8.0) with 100 mM MgCl₂. The assay volume, containing 0.2μM FITC-Aβ1-42 and 0.8 μM Aβ1-42, was 50 μL and the assay temperaturewas 37° C. ADDL assembly was monitored with a Tecan GENios Pro platereader, exciting at a wavelength of 485 nm and detecting emission at awavelength of 515 nm. Kinetic traces were collected by recordingfluorescence intensity and polarization readings every five minutes overa six-hour time course. Negative control reactions, which did notappreciably assemble into ADDLs during this time, lacked MgCl₂ butcontained all other buffer and peptide components. Positive controlreactions contained all buffer components in the absence of addedmonoclonal antibody reagents. To test for ADDL binding and assemblyinhibition, antibodies were incubated with the peptide mixture at eightconcentrations from 500 nM decreasing to 5 nM.

This assay was useful for classifying different profiles of ADDL bindingbehavior and ADDL assembly inhibition. The binding and neutralization oflarger ADDL species, through interaction with ADDL-specific and/orconformational epitopes, serves as a viable therapeutic strategy. Inaddition, the inhibition of oligomerization into large ADDLs by bindingan ADDL-specific and/or conformational epitope present in transient,intermediate ADDL assembly species (non-monomer) provides an alternativestrategy for anti-ADDL therapy. The FP progress curves, whichdemonstrated striking differences between antibodies, denotes suchintermediate or stable species binding. Correlating the FP/FRET behaviorof monoclonal antibodies with other functional, cellular and in vivoeffects allows for the selection of desired immunotherapy modes ofaction.

The results of the analyses disclosed herein indicates that 1F6, 2A10,5F10, 2D6, and 2B4 exhibit potent assembly inhibition, whereas 20C2,1F4, and 4C2 exhibit intermediate assembly inhibition and 2H4, 3B3 and4E2 show weak assembly inhibition (FIG. 4). As summarized in Table 4 andillustrated in FIG. 5, 20C2, 4E2, 3B3 and 5F10 show a variety ofbiochemical behaviors.

TABLE 4 Weak or no Potent Assembly Assembly Inhibition by Inhibition byFP/FRET FP/FRET FP laddering at 30 20C2 4E2 minutes Low or no FP 5F10,1A9 3B3 laddering at 30 minutes

Further, antibody 1A9, one of five purified antibodies (i.e., 1A9, 1E3,1G3, 1A7, and 1E5) generated against a low n-mer-forming peptideAβ1-42-[Nle35-Dpro37], segregates with 5F10 in terms of its assemblyinhibition and FP behavior.

Moreover, 20C2 was found to bind to assemblies of charge-inverted,truncated Aβ7-42 peptide assemblies as determined by SEC/ICC, indicatinga lack of conventional linear epitope binding to the Aβ7-42charge-inverted peptide, which has a very different sequencecorresponding to residues 7-16 of Aβ, i.e., A(7-42)[Orn₇Orn₁₁D₁₃D₁₄E₁₆Nle₃₅]. Therefore, 20C2 binds to conformationalepitopes that depend upon elements from within residues 17-42 of Aβ, butonly when assembled.

EXAMPLE 17 Isolation of Mouse Antibody Variable Region Sequences

The cDNAs coding for the variable domains of the mouse antibody werecloned and sequenced following a polymerase chain reaction (PCR) usingspecially designed primers that hybridize to the 5′-ends of the mouseconstant regions and to the murine leader sequences upstream of the Vregions. This ensured that the mouse variable region sequences obtainedwere complete and accurate. In short, mRNA was extracted from mousehybridoma cell lines using the QIAGEN® OLIGOTEX® Direct mRNA Mini Kitand subsequently converted to cDNA using a first-strand cDNA synthesiskit. The cDNA was then used as template in PCR reactions to obtain theantibody variable region sequences.

To obtain the light chain variable region sequence, eleven independentPCR reactions were set up using each of the eleven light chain 5′ PCRprimers (MKV-1 to MKV-11) and the 3′ PCR primer MKC-1 (Table 5).

TABLE 5 SEQ ID Sequence NO: 5′ Primer MKV-1 GAT CTC TAG A TG AAG ATT GCCTGT TAG GCT GTT GGT GCT G 223 MKV-2 GAT CTC TAG A TG GAG WCA GAC ACA CTCCTG YTA TGG GTG 224 MKV-3 GAT CTC TAG A TG AGT GTG CTC ACT CAG GTC CTGGSG TTG 225 MKV-4 GAT CTC TAG A TG AGG RCC CCT GCT CAG WTT YTT GGM WTCTTG 226 MKV-5 GAT CTC TAG A TG GAT TTW CAG GTG CAG ATT WTC AGC TTC 227MKV-6 GAT CTC TAG A TG AGG TKC YYT GYT SAY CTY CTC TGR GG 228 MKV-7 GATCTC TAG A TG GGC WTC AAA GAT GGA GTC ACA KWY YCW GG 229 MKV-8 GATCTC TAG A TG TGG GGA YCT KTT TYC MMT TTT TCA ATG 230 MKV-9 GAT CTC TAG ATG GTR TCC WCA SCT CAG TTC CTT G 231 MKV-10 GAT CTC TAG A TG TAT ATA TGTTTG TTG TCT ATT TCT 232 MKV-11 GAT CTC TAG A TG GAA GCC CCA GCT CAG CTTCTC TTC C 333 3′ Primer MKC-1 GAT CGA GCT C AC TGG ATG GTG GGA AGA TGG234 Underlined and italic sequences denote XbaI and SacI restrictionsites, respectively. W = A or T, M = A or C, K = G or T, Y = C or T, andR = A or G.

To obtain the heavy chain variable region sequences twelve independentPCR reactions were set up using each of the twelve heavy chain 5′ PCRprimers (MHV-1 to MHV-12) and the appropriate isotype specific 3′ primer(MHCG-1, MHCG-2A, MHCG-2B, MHCG-3) (Table 6).

TABLE 6 SEQ ID Sequence NO: 5′ Primer MHV-1 GAT CTC TAG A TG AAA TGC AGCTGG GGC ATS TTC TTC 235 MHV-2 GAT CTC TAG A TG GGA TGG AGC TRT ATC ATSYTC TT 236 MHV-3 GAT CTC TAG A TG AAG WTG TGG TTA AAC TGG GTT TTT 237MHV-4 GAT CTC TAG A TG RAC TTT GGG YTC AGC TTG RTT T 238 MHV-5 GATCTC TAG A TG GGA CTC CAG GCT TCA ATT TAG TTT TCC TT 239 MHV-6 GATCTC TAG A TG GCT TGT CYT TRG SGC TRC TCT TCT GC 240 MHV-7 GAT CTC TAG ATG GRA TGG AGC KGG RGT CTT TMT CTT 241 MHV-8 GAT CTC TAG A TG AGA GTGCTG ATT CTT TTG TG 242 MHV-9 GAT CTC TAG A TG GMT TGG GTG TGG AMC TTGCTT ATT CCT G 243 MHV-10 GAT CTC TAG A TG GGC AGA CTT ACC ATT CTC ATTCCT G 244 MHV-11 GAT CTC TAG A TG GAT TTT GGG CTG ATT TTT TTT ATT G 245MHV-12 GAT CTC TAG A TG GTG TTA AGT CTT CTG TAC CTG 246 3′ Primer MHCG-1GCATC GAG CTC CAG TGG ATA GAC AGA TGG GGG 247 MHCG-2A GCATC GAG CTC CAGTGG ATA GAC CGA TGG GGG 248 MHCG-2B GCATC GAG CTC CAG TGG ATG AGC TGATGG GGG 249 MHCG-3 GCATC GAG CTC CAA GGG ATA GAC AGA TGG GGC 250Underlined and italic sequences denote XbaI and SacI restriction sites,respectively. W = A or T, M = A or C, K = G or T, Y = C or T, and K = Aor G.

Each of the light chain PCR reactions contained 46 μL INVITROGEN™PLATINUM® PCR Super Mix, 1.0 μL of one of the 100 μM 5′ primers (MKV-1to MKV-11), 1.0 μL of the 100 μM 3′ primer (MKC-1), and 2.0 μL ofhybridoma cDNA. Similar PCR reactions were employed to clone the mouseheavy chain variable region sequences. Reactions were placed in a DNAthermal cycler and, after an initial denaturation step at 97° C. for 2.0minutes, subjected to 30 cycles of: 95° C. for 30 seconds, 55° C. for 45seconds, and 72° C. for 90 seconds. Following the last cycle, a finalextension step at 72° C. for 10 minutes was employed. To determine whichPCR reactions yielded product, 5 μL aliquots from each reaction wereseparated on 1.5% (w/v) agarose/1× TAE buffer gels, containing 0.5 μg/mLethidium bromide. PCR products from reactions that produced fragments ofthe expected size (420 to 500 bp) were then gel purified, digested withXbaI and SacI and ligated into the XbaI and SacI sites in themulticloning region of plasmid pNEB193 (New England Biolabs, Beverly,Mass.). Alternatively, PCR products were ligated directly into plasmidpCR®2.1 using the INVITROGEN™ TA CLONING® kit. Ligation products werethen transformed into XL-1 cells and aliquots of the transformed E. coliwere plated onto LB agar plates containing 50 μg/mL ampicillin andoverlaid with 40 μL of X-Gal stock (50 mg/mL) and 40 μL IPTG (100 mM)solution for blue/white selection. Plates were incubated overnight at37° C. and potential clones were identified as white colonies. DNA fromat least 24 independent clones for each PCR product were sequenced onboth strands using universal forward and reverse primers for pNEB193 andpCR® 2.1. The resulting sequences were then assembled into a contig togenerate a consensus sequence for each antibody light and heavy chainvariable region. Using this approach the sequences were determined forthe light and heavy antibody variable regions of hybridoma's 20C2, 5F10,2D6, 2B4, 4E2, 2H4, 2A10, 3B3, 1F6, 1F4, 2E12 and 4C2 (FIGS. 6A-6X).

The six complementarity-determining regions (CDRs), which form thestructure complementary to the antigen, are underlined in FIGS. 6A-6X.Upon analysis of the CDRs and corresponding antigen epitopes (Table 2),sequence similarities were observed. Antibodies sharing the 3-8 aminoacid epitope of Aβ1-42 (i.e., 2A10, 4C2, 2D6, 4E2, 20C2, 2B4, and 5F10)shared highly homologous CDR1 (FIG. 7A) and CDR2 (FIG. 7B) sequences ofthe heavy chain. Antibody 2H4, which was found to recognize the 1-8amino acid epitope of Aβ1-42, appeared to have unique CDR3 (FIG. 7C)sequences of the heavy chain and unique CDR1 (FIG. 7D), CDR2 (FIG. 7E),and CDR3 (FIG. 7F) sequences of the light chain. Similarly, antibody2E12, which was found to recognize the 3-10 amino acid epitope ofAβ1-42, had unique CDR3 sequences of the heavy chain (FIG. 7C). Further,antibodies 2A10, 2B4, 4C2 and 4E2, having similar affinities for SECPeak 1 and Peak 2 ADDLs (see FIG. 3), shared highly homologous CDR3sequences of the heavy chain (FIG. 7C). Moreover, amino acidsubstitutions in CDR3 of the heavy chain of antibody 4E2 appeared toenhance blockage of binding of ADDLs to neuronal cells, as 4E2 is moreeffective than antibody 2D6 at blocking ADDL binding to neurons and thesequences of the heavy and light chains of 4E2 and 2D6 were identicalexcept for three amino acid residues of CDR3 of the heavy chain; Ser vs.Asn, Thr vs. Ser, and Ile vs. Val for 2D6 and 4E2, respectively (FIG.7C).

EXAMPLE 18 Humanization of Mouse Anti-ADDL Antibody Variable RegionSequences

Mouse antibody heavy and light variable domains nucleic acids obtainedfrom mouse hybridoma cell lines 20C2, 26D6, 4E2, 3B3, 2H4 and 1F6 werehumanized using a CDR grafting approach and in the case of 20C2 and 26D6a veneering strategy. It will be appreciated by those skilled in the artthat humanization of mouse antibody sequences can maximize thetherapeutic potential of an antibody by improving its serum half-lifeand Fc effector functions thereby reducing the anti-globulin response.

Humanization by CDR grafting was carried out by selecting the humanlight and heavy chain variable regions from the NCBI protein databasewith the highest homology to the mouse variable domains. The mousevariable region sequences were compared to all human variable regionsequences in the database using the protein-protein Basic LocalAlignment Search Tool (BLAST). Subsequently, mouse CDRs were joined tothe human framework regions and the preliminary amino acid sequence wasanalyzed. All differences between the mouse and human sequences in theframework regions were evaluated particularly if they were part of thecanonical sequences for loop structure or were residues located at theVL/VH interface (O'Brien and Jones (2001) In: Antibody Engineering,Kontermann and Dubel (Eds.), Springer Laboratory Manuals). Frameworkregions were also scanned for unusual or rare amino acids in comparisonto the consensus sequences for the human subgroup and for potentialglycosylation sites. Wherein amino acid sequence differences existedbetween the mouse and human framework region sequences that were notfound to be involved in canonical sequences, or located at the VL/VHinterface, the human residue was selected at that position. Wherein adifference in a key residue existed, two versions of the variable regionsequence were generated for evaluation. The CDR grafting strategy madethe minimum number of changes to the human framework region so that goodantigen binding was achieved while maintaining human framework regionsthat closely matched the sequence from a natural human antibody. Thedesign of humanized amino acid sequences using CDR grafting is shown inFIG. 8.

Humanized sequences for 20C2 and 26D6 were also designed using aveneering strategy (See, e.g., U.S. Pat. No. 6,797,492). Humanizationwas carried out by selecting the human light and heavy chain variableregions from the NCBI protein database with the highest homology to themouse variable domains, as well as to the closest human antibodygermline family or families (see, Kabat, et al. (1991) Sequences ofproteins of immunological interest, 5^(th) ed., U.S. Dept. Health andHuman Services, NIH, Washington D.C.). The mouse variable regionsequences were compared to all human variable region sequences in thedatabase using protein-protein BLAST. The murine variable sequences andtheir closest human homologues were modeled to the closest crystallizedhuman antibody as determined by computer modeling as practiced in theart. From the model of the murine VH and VL sequences, a surface areamap was constructed, which dictated the solvent accessibility of theamino acids in the mouse heavy and light variable regions. To confirmthe modeling, these exposed residues were compared position-by-positionwith known surface accessible residues (see, e.g., Padlan (1994) Mol.Immunol. 31(3):169-217). A score was assigned for each residue in thesequence designating it as exposed, mostly exposed, partly buried,mostly buried and buried according to established methods (see, U.S.Pat. No. 6,797,492). Mouse framework residues that scored as exposed ormostly exposed and differed from the homologous human sequence werechanged to the human residue at that position. The designed veneeredsequences retained the mouse CDRs, residues neighboring the CDRs,residues known be involved in canonical sequences, residues located atthe VL/VH interface, and residues at the N-terminal sequences of themouse heavy and light chain. The N-terminal sequences are known to becontiguous with the CDR surface and are potentially involved in ligandbinding. Likewise, care was taken to limit changes in Pro, Gly, orcharged residues. Once the veneered sequences were finalized they wereremodeled to look for are any potential obvious structural issues. Insome instances, more then one veneered sequence was generated foranalysis. The design of humanized amino acid sequences using theveneering approach is shown in FIG. 9.

Once the humanized amino acid sequences were selected the sequences werereverse-translated to obtain the corresponding DNA sequence. The DNAsequences were codon-optimized using art-established methods (Lathe(1985) J. Mol. Biol. 183(1):1-12) and designed with flanking restrictionenzyme sites for cloning into human antibody expression vectors. The DNAsequences synthesized are presented in FIGS. 10A-10S. For the 20C2humanized antibodies designed by CDR grafting and veneering, both humanIgG1/kappa and IgG2 m4/kappa versions were constructed, wherein IgG2 m4represents selective incorporation of human IgG4 sequences into astandard human IgG2 constant region. IgG1/kappa and IgG2 m4/kappaversions were also made for the 26D6 CDR grafted antibody. For all otherantibodies only the IgG1/kappa versions were made. The complete aminoacid sequence of the resulting antibodies is shown in FIGS. 11A-11Y.

Antibodies were expressed by co-transient transfection of separate lightand heavy chain expression plasmids into 293 EBNA cells. In cases wheremore then one humanized heavy or light chain sequence was designed for agiven antibody, all combinations of heavy and light chains were combinedto generate the corresponding antibodies. Antibodies were purified fromculture supernatant 7-10 days post-transfection using protein A columnsand used in subsequent analysis.

EXAMPLE 19 Generation of IgG2 m4 Antibodies

IgG2 m4 antibody derivatives were prepared to decrease Fc receptorengagement, C1q binding, unwanted cytotoxicity or immunocomplexformation while maintaining both the long half-life and pharmacokineticproperties of a typical human antibody. The basic antibody format ofIgG2 m4 is that of IgG2, which has been shown to possess a superiorhalf-life in experimental models (Zuckier, et al. (1994) Cancer Suppl.73:794-799). The structure of IgG2 was modified to eliminate C1qbinding, through selective incorporation of IgG4 sequences, whilemaintaining the typical low level of FcγR binding (Canfield and Morrison(1991) J. Exp. Med. 173:1483-1491). This was achieved by usingcross-over points wherein sequences of IgG2 and IgG4 were identical,thereby producing an antibody containing natural Fc sequences ratherthan any artificial mutational sequences.

The IgG2m4 form of the human antibody constant region was formed byselective incorporation of human IgG4 sequences into a standard humanIgG2 constant region, as shown in FIG. 12. Conceptually, IgG2m4 resultedfrom a pair of chain-swaps within the CH2 domain as shown in FIG. 12.Four single mutations were made corresponding to sequences from IgG4.The Fc residues mutated in IgG2 included His268Gln, Val309Leu,Ala330Ser, and Pro331Ser, which minimized the potential for neoepitopes.The specific IgG4 amino acid residues placed into the IgG2 constantregion are shown in Table 7, along with other alternatives from thebasic structure.

TABLE 7 Residue Alternative (Kabat number- Residue Residue Residueresidue in ing) in IgG2 in IgG4 in IgG2m4 IgG2m4 Comment 189 Pro or ProThr Pro Key polymorphism of IgG2; Thr* Pro residue present in IGHG*01allotype and Thr residue present in IGHG2*02 allotype^(a,b). 268 His GlnGln — Change in the B/C loop known to be involved in FcγRII binding^(c).309 Val Leu or Leu Val FcRn binding domain Val 330 Ala Ser Ser — Keyresidue for C1q binding^(d); also potentially involved in binding FcγRIIand FcγRIII^(e). 331 Pro Ser Ser — Key residue for C1q binding^(d,f) andFcγRI binding^(g); also potentially involved in binding FcγRII andFcγRIII^(e). 397 Met or Val Met Val Val residue present in Val* IGHG*01allotype and Met residue present in IGHG2*02 allotype^(a). *Positionsmarked with an asterisk are subject to allelic variations. ^(a)Hougs, etal. (2001) Immunogenetics 52 (3-4): 242-8. ^(b)WO 97/11971.^(c)Medgyesi, et al. (2004) Eur. J. Immunol. 34: 1127-1135. ^(d)Tao, etal. (1991) J. Exp. Med. 173: 1025-1028. ^(e)Armour, et al. (1999) Eur.J. Immunol. 29: 2613. ^(f)Xu, et al. (1994) J. Biol. Chem. 269:3469-3474. ^(g)Canfield and Morrison (1991) J. Exp. Med. 173: 1483.

EXAMPLE 20 Binding Affinity of Humanized Anti-ADDL Antibodies

To evaluate ADDL binding affinity of the humanized antibodies, titrationELISAs were conducted as disclosed herein. Streptavidin-coated, 96-wellmicrotiter plates (Sigma, St. Louis, Mo.) were coated with 10%biotinylated ADDL antigen (1 μM). A series of 2-fold dilutions ofpurified antibody, starting at 500 ng/mL was added to the ADDL capturedplates and the plates were incubated for 2 hours at 25° C. After washingfive times with PBS solution using a plate washer (Bio-Tek, Winooski,Va.), polyclonal goat anti-human kappa light chain antibody (Biomeda,Foster City, Calif.) was added at a 1/2000 dilution in 3% non-fat milkblocker and incubated at room temperature for 1 hour. A rabbit anti-goatIgG (H+L) HRP-conjugated (Bethyl Laboratories, Inc., Montgomery, Tex.)detection antibody was then added at a 1/2000 dilution in blockingsolution and incubated for 1 hour at room temperature. After washingwith PBS, HRP substrate, 3,3′,5′5-tetramethylbenzidine (ready-to-useTMB; Sigma, St. Louis, Mo.) was added and the reaction was stopped after10 minutes with 0.5 N H₂SO₄. Absorbance at wavelength of 450 nm was readin a plate reader (model VICTOR V; Perkin Elmer, Boston, Mass.) and datawere processed using EXCEL® work sheet. Assay variations between plateswere estimated within 20%.

Different groups of humanized antibodies were compared in differentexperiments. A comparison of IgG1 antibodies 20C2A, 20C2B, 3B3, 4E2, 1F6and 2H4 humanized by CDR grafting indicated that all antibodies couldbind to ADDLs, wherein binding with 1F6 was weaker than the majority and20C2A was the strongest. The four different humanized versions of 20C2IgG1 antibodies (two CDR grafted versions and two veneered versions)were also compared and found to exhibit very similar ADDL binding curveswith all binding slightly better then a chimeric 20C2 antibody. Theseven different humanized versions of 26D6 IgG1 (one CDR graftedversions and six veneered versions) were also compared. All were foundto have ADDL binding curves similar to the chimeric form of 26D6. TheIgG1 and IgG2m4 antibodies for the two 20C2 versions humanized by CDRgrafting were also analyzed and found to have comparable binding curvesas did the IgG1 and IgG2m4 isotypes of 26D6 humanized by CDR grafting.

EXAMPLE 21 Inhibition of ADDL Binding to Neurons Using HumanizedAnti-ADDL Antibodies

The humanized anti-ADDL antibodies were further evaluated for theirability to block ADDL binding to primary hippocampal neurons using themethods disclosed herein. The relevant antibodies, or PBS as a control,were mixed at a 1:1 (B103 neuroblastoma cells) or 1:5 (primaryhippocampal neurons) molar ratio with 2.5-10 μm (final concentration) ofbADDLs and incubated for one hour at 37° C. on a slow rotator. After thepreincubation, the antibody/bADDL preparations were added to the B103 orprimary neuron cultures and incubated for an additional hour at 37° C.At the end of the incubation period, the bADDLs/antibody mixture wasremoved and the plates washed six times with media. The cells were thenfixed in 4% paraformaldehyde for ten minutes at room temperature, thesolution removed, fresh fixative added, and the cells fixed for anadditional ten minutes. The cells were permeabilized with 4%paraformaldehyde containing 0.1% TRITON™ X-100 (2 times, each for tenminutes at room temperature), washed six times in PBS and then treatedwith 10% BSA in PBS for one hour at 37° C. Alkalinephosphatase-conjugated streptavidin (1:1,500 in 1% BSA; MolecularProbes, Eugene, Oreg.) was then added to the cells for one hour at roomtemperature. The cells were rinsed six times with PBS, the alkalinephosphatase substrate (CDP-STAR® with SAPPHIRE-II™; Applied Biosystems,Foster City, Calif.) added to the cells and incubated for thirty minutesprior to determining the luminescence on a LJL Luminometer (Analyst AD;LJL Biosystems, Sunnyvale, Calif.). As with the murine antibodies, thehumanized versions of 26D6, 20C2, 4E2, 3B3, 2H4 and 1F6 were capable ofinhibiting the binding of ADDL preparations to B103 neuroblastoma cellsand to primary neurons.

EXAMPLE 22 Affinity Maturation of a Humanized Anti-ADDL Antibody

Nucleic acid molecules encoding humanized 20C2 version A variable heavychain only, light chain only, or heavy chain and light chain togetherwere cloned in the Fab phage-display vector pFab3d. Nucleic acidsequence analysis confirmed sequence and orientation in pFab3d. Theannotated 20C2 Fab sequences in pFab3d are presented in FIG. 13 and setforth herein as SEQ ID NO:255 for the heavy chain and SEQ ID NO:256 forthe light chain. The three constructs were used in the 20C2 maturationprogram using art-established phage-displayed Fab library methods.

Briefly, two libraries were designed to mutate the nine wild-type aminoacids of CDR3 of the light (kappa) chain of 20C2 (i.e.,Phe-Gln-Gly-Ser-Leu-Val-Pro-Leu-Thr; SEQ ID NO:60). These libraries weredesignated LC3-1 and LC3-2 representing light chain CDR3 sequences ofXaa-Xaa-Xaa-Xaa-Xaa-Val-Pro-Leu-Thr (SEQ ID NO:257) andPhe-Gln-Gly-Ser-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:258), respectively.Biotinylated reverse primers, 20C2LC3-1 (SEQ ID NO:259) and 20C2LC3-2(SEQ ID NO:260), were used in combination with forward primer 20C2LC3F(SEQ ID NO:261) to generate the LC3-1 and LC3-2 libraries (see FIG. 14).Primers were purified by polyacrylamide gel electrophoresis, whereas thevector DNA was purified by gel electrophoresis and electroelution. Thetwo light chain libraries were designed to be randomly mutated. Thefinal diversities of the three 10G5H6 LC₃ libraries were 4.76×10⁸ and7.45×10⁸, respectively (Table 8). Sequence analysis of approximately 100clones from the libraries showed 100% diversity of mutant clones at thedesigned amino acid positions.

TABLE 8 20C2 Library Characteristic LC3-1 LC3-2 Vector pFab3d20C2HSpFab3d20C2HS Number of 4.76 × 10⁸ 7.45 × 10⁸ Transformants LibraryDiversity 4.76 × 10⁸ × 0.89 = 7.45 × 10⁸ × 0.90 = 4.24 10⁸ 6.71 10⁸Primary Library 2 mL 2 mL Volume Primary Library 2.13 × 10¹¹ *9.3 × 10¹⁰Titer *Higher titers are achieved by concentration or phage rescue.

Soluble panning of the two 20C2 light chain libraries against highmolecular weight bADDL was completed. Briefly, four rounds of panningwere carried out using biotinylated high molecular weight ADDL (bADDL).The first three rounds were carried out using approximately 1.5 μMantigen concentration (input=1×10¹⁰ to 1×10¹¹). Upon completion of thethird round, the outputs of the two libraries were combined and dividedinto three groups for analysis with 10 nM, 100 nM and approximately 1.5μM antigen to increase panning stringency. As such, a total of 58 outputplates were tested in phage ELISA assays, i.e., two plates per libraryin the first round (a total of four plates), six plates per library inthe second round (a total of 12 plates), eight plates for LC3-1 and 10plates for LC3-2 libraries in the third round (a total of 18 plates) andeight plates for each antigen concentration in the fourth round (a totalof 24 plates).

Panning resulted in 1000 hits, 436 of which were sequenced (Table 9).

TABLE 9 ELISA Round Antigen Input Output % Recovery Screen* Sequenced1^(a) 1.6 μM 2.13 × 10¹⁰  7.3 × 10⁴ 3.42 × 10⁻⁶  0% 0  (0/176) 2^(a) 2.0μM 1.55 × 10¹¹ 1.88 × 10⁵ 1.21 × 10⁻⁶ 1.5%  8  (8/528) 3^(a) 1.1 μM 1.80× 10¹⁰  7.8 × 10⁴  4.3 × 10⁻⁶ 5.8%  41  (41/704) 1^(b) 1.6 μM 9.30 × 10⁹ 5.7 × 10⁴ 6.13 × 10⁻⁶ 2.3%  4  (7/176) 2^(b) 2.0 μM 1.23 × 10¹¹ 1.07 ×10⁵  8.7 × 10⁻⁷ 4.5%  24  (24/528) 3^(b) 1.1 μM 1.37 × 10¹⁰ 3.32 × 10⁵2.42 × 10⁻⁵ 15% 134 (134/880) 4^(c) 1.1 μM  3.0 × 10¹¹ 1.37 × 10⁵  4.6 ×10⁻⁷ 39% — (274/704) 4^(c) 100 nM  3.0 × 10¹¹ 3.88 × 10⁵ 1.29 × 10⁻⁶ 41%— (290/704) 4^(c) 10 nM  3.0 × 10¹¹  1.6 × 10⁵  5.3 × 10⁻⁷ 32% (225/704)225 Total 1000/5104 436 ^(a)20C2 LC3-1 versus high molecular weight 10%bADDL. ^(b)20C2 LC3-2 versus high molecular weight 10% bADDL. ^(c)20C2LC3-1 + 20C2 LC3-2 versus high molecular weight 10% bADDL. *Hits pertotal number of colonies.

Sequence and frequency of highly enriched clones are presented in Table10.

TABLE 10 SEQ Clone ID Round Round Round Designation CDR3 NO: 2 3 4 TotalHu20C2LC FQGSLVPLT 60 6 15 14 35 SJ-p1-31 ADTTHVPLT 262 1 2 3 SJ-p1-14AHSTFVPLT 263 1 1 2 4 4P2-12-E3 AQASFVPLT 264 2 2 SJ-p1-38 AQATKVPLT 2651 1 2 4P3-59 AQSSKVPLT 266 2 2 SJ-p2-14 AQSTLVPLT 267 1 2 3 4P3-11FAASSVPLT 268 2 2 4P3-1 FESTYVPLT 269 2 2 SJ-p2-10 FESSRVPLT 270 1 1 2SJ-p2-11 FNATWVPLT 271 2 2 SJ-p2-60 FQASRVPLT 272 1 5 6 SJ-p1-18FQATRVPLT 273 1 5 6 SJ-p3-51 FQGSFIGLS 274 1 1 2 SJ-p3-16 FQGSFIPGT 2752 3 5 SJ-p8-8F FQGSFLPPS 276 1 1 2 SJ-p3-26 FQGSFLPQL 277 1 2 3 SJ-p3-15FQGSLFPPV 278 1 2 3 SJ-p2-70 FQGSLFSPS 279 1 5 6 SJ-p3-24 FQGSRIPIS 2801 1 2 SJ-p3-33 FQGSRLPVS 281 2 3 5 SJ-p3-14 FQGSRVPLV 282 2 1 3 SJ-p2-1FFQSSFVPLT 283 6 8 14 4P1-22 FQSSRVPLT 284 15 15 SJ-p2-44 GQTTLVPLT 285 13 4 SJ-p1-56 HESTLVPLT 286 2 1 3 4P1-40 HQSSKVPLT 287 4 4 SJ-p2-20IQTSLVPLT 288 2 2 SJ-p1-41 IQAALVPLT 289 1 1 2 SJ-p2-13 LQSSFVPLT 290 14 5 4P1-26 LETSRVPLT 291 3 3 SJ-p1-33 LASSHVPLT 292 2 1 3 SJ-p2-27LNSTTVPLT 293 2 4 6 SJ-p2-62 LQSKSVPLT 294 2 2 4P2-26-E5 LQSVRVPLT 295 33 4P1-32 LQSSLVPLT 296 5 5 SJ-p2-37 LQTGRVPLT 297 2 2 4 SJ-p2-64LQTSFVPLT 298 3 3 4P1-20 LQTSNVPLT 299 5 5 SJ-p2-39 LQTTRVPLT 300 2 6 8SJ-p2-52 LSSTFVPLT 301 3 1 4 SJ-p2-6L LSSTHVPLT 302 2 1 3 4P1-77LTSSAVPLT 303 2 2 SJ-p1-59 LVSSLVPLT 304 2 2 SJ-p2-23 METANVPLT 305 2 2SJ-p1-9M MQSSFVPLT 306 1 3 4 SJ-p2-28 MQSSLVPLT 307 1 2 3 SJ-p1-21MQTSKVPLT 308 1 1 2 4P1-17 SQARMVPLT 309 3 3 SJ-p2-66 SQASRVPLT 310 1 23 SJ-p1-49 TQSTQVPLT 311 2 1 3 SJ-p2-24 VCATFVPLT 312 1 1 2 4P1-41VQSSAVPLT 313 2 2 SJ-p2-51 VQTSLVPLT 314 12 31 43 4P1-64 VQTSVVPLT 315 33 SJ-p2-55 VQTTAVPLT 316 2 2 SJ-p1-25 LQTARVPLT 317 1 3 4

Fab fragments from the 10 top clones based on enrichment frequency wereprepared and a total of 15 clones were converted into IgG1 humanized Aversion and two clones, 20C2-6 and 20C2-8, were converted to IgG1humanized B version. KD values for these clones were measured byBIACORE™ using biotin-Aβ1-20 (Table 11) and bADDL (Table 12) asantigens. Dramatic improvements in affinity were observed as compared toparental humanized 20C2A and 20C2B, as well as mouse 20C2 antibodies. Inparticular, low nanomolar to sub-picomolar KDs were achieved with alight chain CDR3 of the sequence Xaa₁-Gln-Xaa₂-Thr-Arg-Val-Pro-Leu-Thr(SEQ ID NO:318), wherein Xaa₁ is Phe or Leu, and Xaa₁ is Ala or Thr.Moreover, a comparison between KD values obtained with BIACORE™ usingbiotin-Aβ1-20 and bADDL further demonstrates that anti-ADDL antibodiessuch as 20C2 preferentially bind multi-dimensional conformations ofADDLs over monomeric Aβ peptides.

TABLE 11 SEQ ID KD (Biotin-Aβ1-20) Name Clone LC-CDR3 NO: Fab IgG1 #1IgG1 #2 20C2-1A SJ-p2-60 FQASRVPLT 262 91 nM 1.2 nM — 20C2-2A SJ-p1-18FQATRVPLT 273 28 nM 686 pM 2 nM 20C2-3A SJ-p3-16 FQGSFIPGT 275 — 1.7 nM— 20C2-5A SJ-p2-1F FQSSFVPLT 283 41 nM 912 pM 1.5 nM 20C2-6A 4P1-22FQSSRVPLT 284 18 nM 544 pM 714 pM 20C2-6B 4P1-22 FQSSRVPLT 284 — 53 pM —20C2-7A SJ-p2-27 LNSTTVPLT 293 128 nM — — 20C2-8A SJ-p2-39 LQTTRVPLT 30014 nM 140 pM 376 pM 20C2-8B SJ-p2-39 LQTTRVPLT 300 — 46 pM 64 pM 20C2-9ASJ-p2-51 VQTSLVPLT 314 36 nM 241 pM 420 pM 20C2-10A SJ-p3-33 FQGSRLPVS281 — 84 nM — 20C2-11A SJ-p3-6 FQGSLLPLS 319 — — — 20C2-12A 4P1-32LQSSLVPLT 296 617 nM 1.5 nM — 20C2-13A 4p1-20 LQTSNVPLT 299 94 nM 3 nM —20C2-18A SJ-p1-9M MQSSFVPLT 306 126 nM 1.8 nM — 20C2-20A SJ-p3-15FQGSLFPPV 278 21 nM 20C2-22A SJ-p2-66 SQASRVPLT 310 2.3 nM 20C2-23A4P1-40 HQSSKVPLT 287 649 pM 1.5 nM 20C2-24A SJ-p2-44 GQTTLVPLT 285 1.9nM 20C2A FQGSLVPLT 60 27 nM 20C2B FQGSLVPLT 60 5.4 nM Mouse- FQGSLVPLT60 83 nM 3.4 nM 20C2

TABLE 12 SEQ ID KD (bADDL) Name Clone LC-CDR3 NO: Fab IgG1 #1 IgG1 #220C2-1A SJ-p2-60 FQASRVPLT 262 85 nM 75 nM — 20C2-2A SJ-p1-18 FQATRVPLT273 28 nM 15 pM 0.3 pM 20C2-3A SJ-p3-16 FQGSFIPGT 275 — 3.7 nM — 20C2-5ASJ-p2-1F FQSSFVPLT 283 41 nM 317 pM 68 pM 20C2-6A 4P1-22 FQSSRVPLT 28442 nM 4.3 pM 24 pM 20C2-6B 4P1-22 FQSSRVPLT 284 — 53 pM — 20C2-7ASJ-p2-27 LNSTTVPLT 293 435 nM — — 20C2-8A SJ-p2-39 LQTTRVPLT 300 13 nM 3pM 0.7 pM 20C2-8B SJ-p2-39 LQTTRVPLT 300 — 13 pM 0.8 pM 20C2-9A SJ-p2-51VQTSLVPLT 314 40 nM — 2 pM 20C2-10A SJ-p3-33 FQGSRLPVS 281 — 7.7 nM20C2-11A SJ-p3-6 FQGSLLPLS 319 — — — 20C2-12A 4P1-32 LQSSLVPLT 296 238nM 15 pM — 20C2-13A 4p1-20 LQTSNVPLT 299 567 nM 764 nM 20C2-18A SJ-p1-9MMQSSFVPLT 306 85 nM 149 pM 20C2-20A SJ-p3-15 FQGSLFPPV 278 6.9 nM20C2-22A SJ-p2-66 SQASRVPLT 310 198 pM 20C2-23A 4P1-40 HQSSKVPLT 287 85pM 66 pM 20C2-24A SJ-p2-44 GQTTLVPLT 285 114 pM 20C2A FQGSLVPLT 60 20C2BFQGSLVPLT 60 Mouse- FQGSLVPLT 60 62 nM 4.1 nM 20C2

1. An isolated antibody, or an antigen binding fragment of the antibody,that binds amyloid β-derived diffusible ligands comprising: (a) a lightchain variable region comprising, (i) a CDR1 having the sequenceArg-Ser-Ser-Gln-Ser-Xaa₁-Xaa₂-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Xaa₃ (SEQID NO:52), wherein Xaa₁ and Xaa₂ are independently Ala, Val, Leu, Ile,or Pro, and Xaa₃ is Asp, Glu, Arg, His, or Lys, (ii) a CDR2 having thesequence Lys-Xaa₁-Ser-Asn-Arg-Phe-Xaa₂ (SEQ ID NO:57), wherein Xaa₁ isAla, Val, Leu, Ile, or Pro, and Xaa₂ is Ser or Phe, and (iii) a CDR3having the sequence Xaa₁-Gln-Xaa₂-Xaa₃-Xaa₄-Val-Pro-Xaa₅-Thr (SEQ IDNO:65), wherein Xaa₁ is Ser or Phe, Xaa₂ is Gly, Ser or Thr, Xaa₃ is Seror Thr, Xaa₄ is His, Tyr or Leu, and Xaa₅ is Ala, Val, Leu, Ile, or Pro;and (b) a heavy chain variable region comprising, (i) a CDR1 of SEQ IDNO:28, (ii) a CDR2 having the sequenceTyr-Ile-Xaa₁-Xaa₂-Xaa₃-Ser-Xaa₄-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Gly(SEQ ID NO:37), wherein Xaa₁ and Xaa₂ are independently Arg, Ser, Gly,Thr, Cys, Tyr, Asn, Gln, Lys, or His, Xaa₃ is Gly or Val, and Xaa₄ isGly, Ser, Thr, Cys, Tyr, Asn, or Gln, and (iii) a CDR3 of SEQ ID NO:48.2. The isolated antibody of claim 1, or antigen binding fragment of theantibody, wherein (a) the light chain variable region comprises, (i) aCDR1 of SEQ ID NO: 49, (ii) a CDR2 of SEQ ID NO: 56, and (iii) a CDR3 ofSEQ ID NO: 64; and (b) the heavy chain variable region comprises, (i) aCDR1 of SEQ ID NO: 28, (ii) a CDR2 of SEQ ID NO: 36, and (iii) a CDR3 ofSEQ ID NO:
 48. 3. The isolated antibody of claim 1, wherein the heavychain variable region of said antibody comprises SEQ ID NO:92 or SEQ IDNO:95 and the light chain variable region of said antibody comprises SEQID NO:96 or SEQ ID NO:99.
 4. The isolated antibody of claim 1, furthercomprising a heavy chain constant region of SEQ ID NO:254.
 5. Theisolated antibody of claim 1, wherein the antibody is a monoclonalantibody.
 6. A pharmaceutical composition comprising the antibody orantigen binding fragment of claim 1 in admixture with a pharmaceuticallyacceptable carrier.
 7. A kit for detecting Aβ-derived diffusible ligandscomprising the antibody or antigen binding fragment of claim
 1. 8. Amethod for attenuating binding of Aβ-derived diffusible ligands to aneuron comprising contacting the neuron with the antibody or antigenbinding fragment of claim 1 so that binding of Aβ-derived diffusibleligands to the neuron is attenuated.
 9. A method for inhibiting assemblyof Aβ-derived diffusible ligands comprising contacting a samplecontaining amyloid β 1-42 peptides with the antibody or antigen bindingfragment of claim 1 thereby inhibiting assembly of Aβ-derived diffusibleligands.
 10. A method for inhibiting the phosphorylation of tau proteinat Ser202/Thr205 comprising contacting a sample containing a tau proteinwith the antibody or antigen binding fragment of claim 1 therebyinhibiting the phosphorylation of tau protein at Ser202/Thr205.
 11. Amethod for attenuating the symptoms of a disease associated withAβ-derived diffusible ligands comprising administering an effectiveamount of the pharmaceutical composition of claim
 6. 12. A method foridentifying a putative therapeutic agent that attenuates the binding ofAβ-derived diffusible ligands to neurons comprising (a) contacting acomposition comprising a neuron with Aβ-derived diffusible ligands inthe presence of an agent; (b) contacting the composition with theantibody or antigen binding fragment of claim 1; and (c) detecting theamount of antibody or antigen binding fragment bound to the neuron inthe presence of the agent, wherein a decrease in the amount of antibodyor antigen binding fragment bound in the presence of the agent ascompared to the amount of antibody bound in the absence of the agentindicates that the agent is a putative therapeutic agent for attenuatingbinding of Aβ-derived diffusible ligands to neurons.
 13. A method fordetecting Aβ-derived diffusible ligands in a sample comprisingcontacting a sample with the antibody or antigen binding fragment ofclaim 1 and determining the presence of a complex comprising theAβ-derived diffusible ligands and antibody or antigen binding fragment.14. A method for diagnosing a disease associated with Aβ-deriveddiffusible ligands comprising contacting a biological sample with theantibody or antigen binding fragment of claim 1 and determining thepresence of a complex comprising the Aβ-derived diffusible ligands andantibody or antigen binding fragment wherein the presence of the complexis diagnostic of a disease associated with Aβ-derived diffusibleligands.